Detection of oligosaccharides

ABSTRACT

Provided herein are processes for detecting oligosaccharides in a biological sample. In specific instances, the biological sample is provided from an individual suffering from a disorder associated with abnormal glycosaminoglycan accumulation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 14/706,794, filed May 7, 2015, which is a continuation of U.S.application Ser. No. 12/649,094, filed Dec. 29, 2009 (now U.S. Pat. No.9,029,530, issued May 12, 2015), which claims the benefit of U.S.Provisional Application No. 61/164,365, filed Mar. 27, 2009, and U.S.Provisional Application No. 61/142,291, filed Jan. 2, 2009, thedisclosures of each of which are incorporated by reference herein intheir entireties.

BACKGROUND OF THE INVENTION

Glycosaminoglycans comprise a reducing end and a non-reducing end.Normal biological processes degrade glycosaminoglycans (such as heparansulfate which has a normal component of about 50-80 kDa) intomonosaccharides. Disorders associated with abnormal glycosaminoglycandegradation, biosynthesis, and/or accumulation can result in anaccumulation of glycosaminoglycans and fragments thereof.

SUMMARY OF THE INVENTION

Described herein are populations of glycosaminoglycans that aretransformed into populations of oligosaccharides using glycosaminoglycanlyases. Further described herein are the use of analytical instrumentsto characterize the population of oligosaccharides in order to providerelevant information about the population of oligosaccharides, thepopulation of glycosaminoglycans and the biological sample that providedthe population of glycosaminoglycans.

Provided in certain embodiments herein is a process for diagnosing theidentity and/or severity of abnormal glycosaminoglycan accumulation inan individual, or a disorder thereof, the process comprising the stepsof:

-   -   a. using an analytical instrument to detect the presence of        and/or measure the amount of a population of one or more        oligosaccharides present in a transformed biological sample that        has been prepared by:        -   treating a population of glycosaminoglycans, in or isolated            from a biological sample from the individual to transform            the glycosaminoglycans into the population of the one or            more oligosaccharide;    -   b. displaying or recording the presence of or a measure of a        population of one or more oligosaccharide.

In certain embodiments, provided is a process for diagnosing thepresence, identity, and/or severity of abnormal glycosaminoglycanaccumulation in an individual, or a disorder thereof, the processcomprising the steps of:

-   -   a. generating a biomarker comprising of one or more non-reducing        end oligosaccharides, wherein the biomarker is a saturated        oligosaccharide and is generated by treating a population of        glycosaminoglycans, in or isolated from a biological sample from        the individual, with at least one digesting glycosaminoglycan        lyases, wherein prior to lyase treatment, such oligosaccharide        biomarker is not present in abundance in samples from        individuals with abnormal glycosaminoglycan accumulation        relative to individuals with normal glycosaminoglycan;    -   b. using an analytical instrument to detect the presence of        and/or measure the amount of the biomarker produced and        displaying or recording the presence of or a measure of a        population of the biomarker.

In specific embodiments, the presence of and/or measure the amount ofthe biomarker is utilized to diagnose of the presence, identity, and/orseverity of abnormal glycosaminoglycan accumulation.

In certain embodiments, the oligosaccharide(s) detected or measured isone or more C4-C5 non-reducing end saturated oligosaccharide(s).

In some embodiments, treating a population of glycosaminoglycans totransform the glycosaminoglycans into the population of the one or moreoligosaccharide comprises contacting the glycosaminoglycans with atleast one digesting glycosaminoglycan lyase. In some embodiments, the atleast one digesting glycosaminoglycan lyase is one or more heparinlyase, one or more chondroitinase, one or more keratanase, one or morehyaluronidase, or a combination thereof. In specific embodiments, the atleast one digesting glycosaminoglycan lyase is one or more heparinlyase.

In certain embodiments, the one or more oligosaccharides detected and/ormeasured are free of carbon-carbon unsaturation. In various embodiments,the abnormal glycosaminoglycan accumulation comprises abnormal heparansulfate accumulation, abnormal chondroitin sulfate accumulation,abnormal keratan sulfate accumulation, abnormal hyaluronan accumulation,or a combination thereof. In specific embodiments, the abnormalglycosaminoglycan accumulation is abnormal heparan sulfate accumulation.

In various embodiments, any process descried herein of preparing atransformed biological sample comprises purifying a population ofoligosaccharides in the biological sample that has been treated with theat least one heparin lyase, the transformed biological sample comprisingthe isolated population of oligosaccharides. In some embodiments, anyprocess described herein of preparing a transformed biological samplecomprises purifying a population of glycosaminoglycans in the biologicalsample prior to treatment with the at least one heparin lyase.

In certain embodiments, any process described herein of detecting thepresence of or measuring the amount of a population of one or moreoligosaccharide present in a transformed biological sample comprises:

-   -   a. isolating a subpopulation of one or more oligosaccharides in        the transformed biological sample; and    -   b. detecting the presence of and/or measuring the amount of one        or more oligosaccharides present in the subpopulation.

In specific embodiments, a subpopulation of one or more oligosaccharidesis isolated using, by way of non-limiting example, chromatography orelectrophoresis. In specific embodiments, the chromatography is highperformance liquid chromatography (HPLC), gas chromatography (GC),column chromatography, affinity chromatography, or thin layerchromatography (TLC). In some embodiments, any process of detectingoligosaccharides described herein comprises detecting oligosaccharidesusing mass spectrometry.

In some embodiments, any process described herein of preparing atransformed biological sample comprises tagging the reducing end of arepresentative portion of the one or more oligosaccharides in thetransformed biological sample with a detectable label. In specificembodiments, the detectable label is a mass label, a radio label, afluorescent label, a chromophore label, or affinity label. In someembodiments, the tagged portion of the one or more oligosaccharides isdetected or measured using UV-Vis spectroscopy, IR spectroscopy, massspectrometry, or a combination thereof

In certain embodiments, a digesting glycosaminoglycan lyase utilized inany process described herein comprises heparan sulfate, chondroitinsulfate, keratan sulfate, hyaluronan, or a combination thereof. Inspecific embodiments, a digesting glycosaminoglycan lyase utilized inany process described herein comprises heparan sulfate.

In some embodiments, a process described herein comprises detecting ormeasuring a disaccharide having the formula:[IdoA-GlcN(Ac)_(m)](SO₃R)_(n), wherein m is 0-1, n is 0-3, and R is H ora negative charge. In some embodiments, the term R used in any formuladescribed herein is H or a negative charge.

In specific embodiments, any process described herein comprisesdetecting or measuring a disaccharide with the formula:

In some embodiments, the disaccharide above is detected and/or measuredin a process of diagnosing a disorder associated with abnormalglycosaminoglycan degradation that is MPS I.

In some embodiments, any process described herein comprises detecting ormeasuring a disaccharide with the formula:

In some embodiments, the disaccharide above is detected and/or measuredin a process of diagnosing a disorder associated with abnormalglycosaminoglycan degradation that is MPS II.

In some embodiments, any process described herein comprises detecting ormeasuring a trisaccharide with the formula[GlcN(Ac)_(m)-(IdoA/GlcA)-GlcN(Ac)_(n)](SO₃R)_(p), wherein IdoA/GlcA iseither IdoA or GlcA, m is 0-1, n is 0-1, p is 0-5, and R is H or anegative charge.

In certain embodiments, any process described herein comprises detectingor measuring a trisaccharide with the formula:

In some embodiments, the trisaccharide above is detected and/or measuredin a process of diagnosing a disorder associated with abnormalglycosaminoglycan degradation that is MPS IIIA.

In certain embodiments, any process described herein comprises detectingor measuring a trisaccharide with the formula:

In some embodiments, the trisaccharide above is detected and/or measuredin a process of diagnosing a disorder associated with abnormalglycosaminoglycan degradation that is MPS IIIB.

In certain embodiments, any process described herein comprises detectingor measuring a trisaccharide with the formula:

In some embodiments, the trisaccharide above is detected and/or measuredin a process of diagnosing a disorder associated with abnormalglycosaminoglycan degradation that is MPS IIIC.

In certain embodiments, any process described herein comprises detectingor measuring a trisaccharide with the formula:

In some embodiments, the trisaccharide above is detected and/or measuredin a process of diagnosing a disorder associated with abnormalglycosaminoglycan degradation that is MPS IIID

In certain embodiments, any process described herein comprises detectingor measuring a disaccharide with the formula[GlcA-GlcN(Ac)_(n)](SO₃R)_(m), wherein n is 0-1, m is 0-2, and R is H ora negative charge.

In certain embodiments, any process described herein comprises detectingor measuring a

In some embodiments, the disaccharide above is detected and/or measuredin a process of diagnosing a disorder associated with abnormalglycosaminoglycan degradation that is MPS VII.

In various embodiments of the processes described herein, a populationof glycosaminoglycans treated with at least one digestingglycosaminoglycan lyase comprises dermatan sulfate, chondroitin sulfate,or a combination thereof. In certain embodiments, any process describedherein comprises detecting or measuring a disaccharide with the formula:[Ido-GalNAc](SO₃R)_(n), wherein n=0-2, and each R is independently H ora negative charge. In some embodiments, any process described hereincomprises detecting or measuring a trisaccharide with the formula:[GalNAc4S-Ido-GlcNAc](SO₃R)_(m), wherein n=0-3; and/or one or moretrisaccharide with the formula: [GalNAc4S-GlcA-GlcNAc](SO₃R)_(m),wherein m=0-1, and wherein each R is independently H or a negativecharge.

In certain embodiments, any process described herein comprises detectingor measuring a disaccharide with the formula: [Gal6S-GalNAc](SO₃R)_(n),wherein n=0-1, and wherein each R is independently H or a negativecharge. In specific embodiments, detection and/or measurement of[Gal6S-GalNAc](SO₃R)_(n) is used in a method of diagnosing MPS IVA orthe severity thereof. In some embodiments, any process described hereincomprises detecting or measuring a disaccharide with the formula:[Gal-GalNAc](SO₃R)_(n), wherein n=0-1, and wherein each R isindependently H or a negative charge. In specific embodiments, detectionand/or measurement of [Gal-GalNAc](SO₃R)_(n) is used in a method ofdiagnosing MPS IVB or the severity thereof.

In some embodiments, any process described herein comprises:

-   -   a. comparing an amount of a population of one or more        oligosaccharide present in a transformed biological sample to an        amount of a population of one or more oligosaccharide present in        a control biological sample that has been treated in a manner        substantially similar to the transformed biological sample.

In certain embodiments, a control biological sample utilized in anyprocess described herein was provided from an individual that does nothave mucopolysaccharidosis (e.g., a non-MPS cell line). In someembodiments, any control biological sample utilized in a processdescribed herein was provided from an individual that hasmucopolysaccharidosis. In specific embodiments, a control biologicalsample was provided from an individual that has MPS I, MPS II, MPS IIIA,MPS IIIB, MPS IIIC, MPS IIID, MPS IVA, MPS IVB, MPS VII, MPS IX, or acombination thereof. In specific embodiments, a control biologicalsample was provided from an individual that has MPS I, MPS II, MPS IIIA,MPS IIIB, MPS IIIC, MPS IIID, MPS VII, or a combination thereof.

Provided in certain embodiments herein is an analytical samplecomprising any oligosaccharide described herein, including anoligosaccharide described herein and further attached to a detectablelabel (e.g., at the reducing end of the oligosaccharide). In specificembodiments, provided herein is an analytical sample comprising one ormore of any of FIGS. 4-23.

In specific embodiments, an analytical sample provided for herein is foruse in high performance liquid chromatography. In some embodiments, ananalytical sample provided for herein is for use in mass spectrometry.In certain embodiments, an analytical sample provided for herein is foruse in gas chromatography. In some embodiments, any analytical sampleprovided herein comprises at least one disaccharide or trisaccharidefrom a transformed biological sample from an individual with a disorderassociated with abnormal glycosaminoglycan accumulation.

Provided in some embodiments herein is an analytical method comprisingtreating a biological sample that comprises glycosaminoglycans with atleast one digesting glycosaminoglycan lyase to transform arepresentative portion of the glycosaminoglycans into one or moreoligosaccharides. In certain embodiments an analytical method providedfor herein comprises purifying one or more oligosaccharides from othercomponents of the biological sample. In some embodiments, the purifyingstep includes use of chromatography. In various embodiments, ananalytical method provided for herein comprises detecting and/ormeasuring the presence of at least one of the oligosaccharides (e.g.,after purification). In certain embodiments, oligosaccharides aredetected and/or measured according to any process or method (usedinterchangeably herein) described herein using UV-Vis spectroscopy, IRspectroscopy, mass spectrometry, or a combination thereof. In someembodiments, any process described herein comprises tagging at least oneof the oligosaccharides with a detectable label. In certain embodiments,the at least one digesting glycosaminoglycan lyase utilized in anyprocess or method described herein comprises one or more heparin lyase,one or more chondroitinase, one or more keratanase, one or morehyaluronidase, or a combination thereof.

In specific embodiments, an analytical method described herein is usedin a method of detecting and/or measuring one or more oligosaccharidesthat are free of carbon-carbon unsaturation.

In certain embodiments, any process described herein comprises detectingor measuring a disaccharide with the formula:[IdoA-GlcN(Ac)_(m)](SO₃R)_(n), wherein m is 0-1, n is 0-3, and R is H ora negative charge. In some embodiments, any process described hereincomprises detecting or measuring a trisaccharide with the formula:[GlcN(Ac)_(m)-(IdoA/GlcA)-GlcN(Ac)p](SO₃R)_(p), wherein IdoA/GlcA iseither IdoA or GlcA, m is 0-1, n is 0-1, and p is 0-5. In certainembodiments, any process described herein comprises detecting ormeasuring a disaccharide with the formula: [GlcA-GlcN(Ac)p](SO₃R)_(n),wherein n is 0-1, and m is 0-2. In some embodiments, any processdescribed herein comprises detecting or measuring a disaccharide withthe formula: [Ido-GalNAc](SO₃R)_(p), wherein n=0-2, and each R isindependently H or a negative charge. In certain embodiments, anyprocess described herein comprises detecting or measuring atrisaccharide with the formula: [GalNAc4S-Ido-GalNAc](SO₃R)_(n), whereinn=0-3; and/or one or more trisaccharide with the formula:[GalNAc4S-GlcA-GalNAc](SO₃R)_(n), wherein m=0-2, and wherein each R isindependently H or a negative charge. In certain embodiments, anyprocess described herein comprises detecting or measuring a disaccharidewith the formula: GlcA-GlcNAc. In certain embodiments, any processdescribed herein comprises detecting or measuring a disaccharide withthe formula: [GlcA-GalNAc](SO₃R)_(m), wherein m is 0-2 (e.g., fordiagnosing CS accumulation in MPS VII, or the severity thereof). In someembodiments, any process described herein comprises detecting ormeasuring a trisaccharide with the formula:[GlcNAc6S-Gal-GlcNAc](SO₃R)_(n), wherein n=0-2, and wherein each R isindependently H or a negative charge. In certain embodiments, anyprocess described herein comprises detecting or measuring a disaccharidewith the formula: [Gal6S-GlcNAc](SO₃R)_(n), wherein n=0-1, and whereineach R is independently H or a negative charge. In certain embodiments,any process described herein comprises detecting or measuring adisaccharide with the formula: [Gal-GlcNAc](SO₃R)_(n), wherein n=0-1,and wherein each R is independently H or a negative charge.

In certain embodiments, a process described herein includes a method ofmonitoring the treatment of disorders associated with the abnormaldegradation, biosynthesis and/or accumulation of glycosaminoglycans(GAGs), the methods comprising:

-   -   a. following administration of an agent for treating MPS to an        individual in need thereof, generating a biomarker comprising of        one or more non-reducing end oligosaccharides, wherein the        biomarker is a saturated oligosaccharide and is generated by        treating a population of glycosaminoglycans, in or isolated from        a biological sample from the individual, with at least one        digesting glycosaminoglycan lyases, wherein prior to lyase        treatment, such oligosaccharide biomarker is not present in        abundance in samples from individuals with abnormal        glycosaminoglycan accumulation relative to individuals with        normal glycosaminoglycan;    -   b. using an analytical instrument to detect the presence of        and/or measure the amount of the biomarker produced and        displaying or recording the presence of or a measure of a        population of the biomarker.

In specific embodiments, increases or decreases in the amount of thebiomarker measured (e.g., as compared to a biological sample previouslyanalyzed in a similar or identical manner) is utilized to monitor thetreatment of disorders associated with the abnormal degradation,biosynthesis and/or accumulation of glycosaminoglycans.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 illustrates the cleavage of the glycosaminoglycan (GAG) heparansulfate with a glycosaminoglycan lyase (heparinase II).

FIG. 2 illustrates lyase liberation of three classes of di- andtrisaccharides from normal and MPS GAGs. The non-reducing end (NRE)fragments are used as biomarkers in certain embodiments herein.

FIGS. 3A-3C illustrate various oligosaccharide residues of GAGs. FIG. 3Aillustrates the heparan sulfate fragments that accumulate in MPS IIpatients considering only the 3 most common modifications (N-, 2-O, and6-O sulfation). Even for a relatively small nonasaccharides (n=4), thereare well over 1,000 potential structures. FIGS. 3B and 3C illustratepredicted non-reducing end heparan sulfate biomarkers for 7 MPS classes.

FIG. 4 illustrates oligosaccharides of Formula I.

FIG. 5 illustrates oligosaccharides of Formula III.

FIG. 6 illustrates oligosaccharides of Formula IV.

FIG. 7 illustrates oligosaccharides of Formula V.

FIG. 8 illustrates oligosaccharides of Formula VI.

FIG. 9 illustrates oligosaccharides of Formula VII.

FIG. 10 illustrates oligosaccharides of Formula VIII.

FIG. 11 illustrates oligosaccharides of Formula IX.

FIG. 12 illustrates oligosaccharides of Formula X.

FIG. 13 illustrates oligosaccharides of Formula XI.

FIG. 14 illustrates oligosaccharides of Formula XII.

FIG. 15 illustrates oligosaccharides of Formula XIII.

FIG. 16 illustrates oligosaccharides of Formula XIV.

FIG. 17 illustrates oligosaccharides of Formula XV.

FIG. 18 illustrates oligosaccharides of Formula XVIII.

FIG. 19 illustrates oligosaccharides of Formula XVIX.

FIG. 20 illustrates oligosaccharides of Formula XX.

FIG. 21 illustrates oligosaccharides of Formula XXX.

FIG. 22 illustrates oligosaccharides of Formula XXXI.

FIG. 23 illustrates oligosaccharides of Formula XXXII.

FIG. 24 illustrates disaccharides isolated from heparan sulfate from anormal human sample.

FIG. 25 illustrates disaccharides isolated from an MPS I (iduronidasedeficient) sample.

FIG. 26 illustrates the detection of oligosaccharides described hereinin individuals having an MPS disease state as compared to individualslacking an MPS disease state.

FIG. 27 illustrates mass spectrometry results for non-reducing endoligosaccharides in a non-MPS sample.

FIG. 28A illustrates mass spectrometry results for non-reducing endoligosaccharides in a MPS I sample. FIG. 28B is an enlargedrepresentation of the ‘28B’ region of FIG. 28A.

FIG. 29A illustrates mass spectrometry results for non-reducing endoligosaccharides in a MPS II sample. FIG. 29B is an enlargedrepresentation of the ‘29B’ region of FIG. 29A. FIG. 29C is an enlargedrepresentation of the ‘29C’ region of FIG. 29A.

FIG. 30A illustrates mass spectrometry results for non-reducing endoligosaccharides in a MPS IIIA sample. FIG. 30B is an enlargedrepresentation of the ‘30B’ region of FIG. 30A.

FIG. 31A illustrates mass spectrometry results for non-reducing endoligosaccharides in a MPS IIIB sample. FIG. 31B is an enlargedrepresentation of the ‘31B’ region of FIG. 31A. FIG. 31C is an enlargedrepresentation of the ‘31C’ region of FIG. 31A.

FIG. 32 illustrates mass spectrometry results showing the accumulationof GAG non-reducing end trisaccharide residues in the liver of an MPSIIIB mouse.

FIG. 33 illustrates mass spectrometry results showing the accumulationof GAG non-reducing end trisaccharide residues in the brain of an MPSTIM mouse.

FIG. 34 illustrates mass spectrometry results showing the accumulationof GAG non-reducing end trisaccharide residues in the kidney of an MPSIIIB mouse.

FIG. 35 illustrates the detection of MPS disease in urine samples.

FIG. 36 illustrates the detection of MPS disease in serum samples.

FIG. 37 illustrates the detection of MPS disease in CSF samples.

FIG. 38 illustrates the detection of MPS disease in tissue samples.

FIG. 39 illustrates monitoring and detecting the response to therapy inserum from MPS patients.

FIG. 40 illustrates monitoring and detecting MPS disease severity.

FIG. 41 illustrates monitoring and detecting the differential responseto therapy in CSF from MPS patients.

FIG. 42 illustrates monitoring and detecting response to therapy intissue samples from MPS patients.

FIGS. 43A, 43B, and 43C illustrate monitoring and detecting the responseto therapy in serum from individuals suffering from MPS. FIGS. 43A, 43B,and 43C illustrate the treatment of different patients.

FIG. 44 illustrates detecting carriers of the genetic cause of MPS.

DETAILED DESCRIPTION OF THE INVENTION

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

Provided in certain embodiments herein are analytical methods fordetecting and/or identifying glycosaminoglycans (GAGs) or other glycans(e.g., glycolipids) in biological sample. In certain embodiments, theglycans, e.g., glycosaminoglycans (GAGs), are present in cells within abiological sample (e.g., within a lysosome thereof), and/or are presentin a biological sample free of cells. In certain embodiments, providedherein is a method of diagnosing any disorder characterized by theaccumulation of glycosaminoglycans, such as a lysosomal storage disease(LSD). In some embodiments, the glycosaminoglycan accumulation is aprimary accumulative effect. In certain instances, primary accumulativeeffects include accumulation that is a direct result of an abnormalbiosynthetic process, such as abnormal production enzymes involved inthe glycan biosynthetic pathway (e.g., under-production or production ofpoorly functioning enzymes), including glycan bio-synthesis ordepolymerization. In other embodiments, the glycosaminoglycanaccumulation is a secondary accumulative effect. In certain embodiments,a secondary accumulative effect results from a cascading effect, e.g.,accumulation of other components, such as GAGs or other glycans, such asglycolipids, causes the GAG biosynthetic pathway to be hindered orinterrupted.

In certain embodiments, glycosaminoglycans include, by way ofnon-limiting example, heparan sulfate, heparin, chondroitin sulfate,dermatan sulfate, hyaluronan, keratan sulfate, or the like, or acombination thereof. In certain embodiments, an analytical methodprovided herein comprising treating a biological sample that comprisesglycosaminoglycans with at least one agent suitable for cleaving bondsbetween saccharide residues of glycosaminoglycans. In specificembodiments, treating a biological sample that comprisesglycosaminoglycans with at least one agent suitable for cleaving bondsbetween saccharide residues of glycosaminoglycans comprises treating thebiological sample with one or more digesting glycosaminoglycan (GAG)lyase. In some embodiments, any glycosaminoglycan (GAG) lyase suitablefor cleaving the bonds (e.g., the bonds linking saccharide residues ofthe GAG to one another) of a glycosaminoglycan (GAG) analyze isutilized. In some embodiments, the lyase is utilized to transform arepresentative portion of the glycosaminoglycans into one or moreoligosaccharides. In certain embodiments, such glycosaminoglycan (GAG)lyases are suitable for preparing di- and/or tri-saccharides from theglycosaminoglycan present. Glycosaminoglycan (GAG) lyases suitable foruse in various embodiments provided herein include, by way ofnon-limiting example, one or more heparin lyase (heparinase), one ormore chondroitinase, one or more keratanase, one or more hyaluronidase,or a combination thereof. Other glycans that are optionally detected bya method described herein include, e.g., glycolipids.

In some embodiments, lyases utilized herein include, by way ofnon-limiting example, Hyaluronate lyase, Pectate lyase,Poly(beta-D-mannuronate) lyase, Chondroitin ABC lyase, Chondroitin AClyase, Oligogalacturonide lyase, Heparin lyase, Heparin-sulfate lyase,Pectate disaccharide-lyase, Pectin lyase, Poly(alpha-L-guluronate)lyase, Xanthan lyase, Exo-(1->4)-alpha-D-glucan lyase, Glucuronan lyase,Anhydrosialidase, Levan fructotransferase, Inulin fructotransferase,Inulin fructotransferase, Chondroitin B lyase. In certain instances,Hyaluronate lyase (EC 4.2.2.1) is an enzyme that catalyzes the cleavageor hyaluronate chains at a beta-D-GalNAc-(1->4)-beta-D-GlcA bond,ultimately breaking the polysaccharide down to3-(4-deoxy-beta-D-gluc-4-enuronosyl)-N-acetyl-D-glucosamine. In someinstances, Pectate lyase (EC 4.2.2.2) is an enzyme that catalyzes theeliminative cleavage of (1->4)-alpha-D-galacturonan to giveoligosaccharides with 4-deoxy-alpha-D-galact-4-enuronosyl groups attheir non-reducing ends. In certain instances, Poly(beta-D-mannuronate)lyase (EC 4.2.2.3) is an enzyme that catalyzes the eliminative cleavageof polysaccharides containing beta-D-mannuronate residues to giveoligosaccharides with 4-deoxy-alpha-L-erythro-hex-4-enopyranuronosylgroups at their ends. In some instances, Chondroitin ABC lyase (EC4.2.2.4) is an enzyme that catalyzes the eliminative degradation ofpolysaccharides containing 1,4-beta-D-hexosaminyl and1,3-beta-D-glucuronosyl linkages to disaccharides containing4-deoxy-beta-D-gluc-4-enuronosyl groups. In some instances, ChondroitinABC lyase (EC 4.2.2.4) also catalyzes the eliminative cleavage ofdermatan sulfate containing 1,4-beta-D-hexosaminyl and1,3-beta-D-glucurosonyl or 1,3-alpha-L-iduronosyl linkages todisaccharides containing 4-deoxy-beta-D-gluc-4-enuronosyl groups toyield a 4,5-unsaturated dermatan-sulfate disaccharide(deltaUA-GalNAc-4S). In certain instances, Chondroitin AC lyase (EC4.2.2.5) is an enzyme that catalyzes the eliminative degradation ofpolysaccharides containing 1,4-beta-D-hexosaminyl and1,3-beta-D-glucuronosyl linkages to disaccharides containing4-deoxy-beta-D-gluc-4-enuronosyl groups. In some instances,Oligogalacturonide lyase (EC 4.2.2.6) is an enzyme that catalyzes thecleavage of 4-(4-deoxy-beta-D-gluc-4-enuronosyl)-D-galacturonate into 25-dehydro-4-deoxy-D-glucuronate. In certain instances, Heparin lyase (EC4.2.2.7) is an enzyme that catalyzes the eliminative cleavage ofpolysaccharides containing 1,4-linked D-glucuronate or L-iduronateresidues and 1,4-alpha-linked 2-sulfoamino-2-deoxy-6-sulfo-D-glucoseresidues to give oligosaccharides with terminal4-deoxy-alpha-D-gluc-4-enuronosyl groups at their non-reducing ends. Insome instances, Heparin lyase (EC 4.2.2.7) tolerates alternativesulfation of the substrate. In some instances, Heparin-sulfate lyase (EC4.2.2.8) is an enzyme that catalyzes the eliminative cleavage ofpolysaccharides containing 1,4-linked D-glucuronate or L-iduronateresidues and 1,4-alpha-linked 2-sulfoamino-2-deoxy-6-sulfo-D-glucoseresidues to give oligosaccharides with terminal4-deoxy-alpha-D-gluc-4-enuronosyl groups at their non-reducing ends. Insome instances, Heparin-sulfate lyase (EC 4.2.2.8) tolerates alternativesulfation of the substrate. In certain instances, Pectatedisaccharide-lyase (EC 4.2.2.9) is an enzyme that catalyzes theeliminative cleavage of4-(4-deoxy-alpha-D-galact-4-enuronosyl)-D-galacturonate from thereducing end of pectate, i.e. de-esterified pectin. In some instances,Pectin lyase (EC 4.2.2.10) is an enzyme that catalyzes the eliminativecleavage of (1->4)-alpha-D-galacturonan methyl ester to giveoligosaccharides with 4-deoxy-6-O-methyl-alpha-D-galact-4-enuronosylgroups at their non-reducing ends. In certain instances,Poly(alpha-L-guluronate) lyase (EC 4.2.2.11) is an enzyme that catalyzesthe eliminative cleavage of polysaccharides containing a terminalalpha-L-guluronate group, to give oligosaccharides with4-deoxy-alpha-L-erythro-hex-4-enuronosyl groups at their non-reducingends. In some instances, Xanthan lyase (EC 4.2.2.12) is an enzyme thatcatalyzes the cleavage of the beta-D-mannosyl-beta-D-1,4-glucuronosylbond on the polysaccharide xanthan. In certain instances,Exo-(1->4)-alpha-D-glucan lyase (E.C. 4.2.2.13) is an enzyme thatcatalyzes the sequential degradation of (1->4)-alpha-D-glucans from thenon-reducing end with the release of 1,5-anhydro-D-fructose. In someinstances, Glucuronan lyase (EC 4.2.2.14) is an enzyme that catalyzesthe eliminative cleavage of (1->4)-beta-D-glucuronans. This produceseither oligosaccharides with 4-deoxy-beta-D-gluc-4-enuronosyl groups attheir non-reducing ends, or, if the substrate is completely degraded,glucuronans produce tetrasaccharides. In certain instances,Anhydrosialidase (EC 4.2.2.15) is an enzyme that catalyzes theelimination of alpha-sialyl groups in N-acetylneuraminic acidglycosides, releasing 2,7-anhydro-alpha-N-acetylneuraminate. In someinstances, Levan fructotransferase (DFA-IV-forming) (EC 4.2.2.16) is anenzyme that produces di-beta-D-fructofuranose 2,6′: 2′,6-dianhydride(DFA IV) by successively eliminating the diminishing(2->6)-beta-D-fructan (levan) chain from the terminalD-fructosyl-D-fructosyl disaccharide. In certain instances, Inulinfructotransferase (DFA-I-forming) (EC 4.2.2.17) is an enzyme thatproduces alpha-D-fructofuranose beta-D-fructofuranose 1,2′:2,1′-dianhydride (DFA I) by successively eliminating the diminishing(2->1)-beta-D-fructan (inulin) chain from the terminalD-fructosyl-D-fructosyl disaccharide. In some instances, Inulinfructotransferase (DFA-III-forming) (EC 4.2.2.18) is an enzyme thatproduces alpha-D-fructofuranose beta-D-fructofuranose1,2′:2,3′-dianhydride (DFA III) by successively eliminating thediminishing (2->1)-beta-D-fructan (inulin) chain from the terminalD-fructosyl-D-fructosyl disaccharide. In certain instances, ChondroitinB lyase (EC 4.2.2.19) is an enzyme that catalyzes the eliminativecleavage of dermatan sulfate containing 1,4-beta-D-hexosaminyl and1,3-beta-D-glucurosonyl or 1,3-alpha-L-iduronosyl linkages todisaccharides containing 4-deoxy-beta-D-gluc-4-enuronosyl groups toyield a 4,5-unsaturated dermatan-sulfate disaccharide(deltaUA-GalNAc-4S). Any other suitable enzyme is also optionallyutilized. For example, any keratanase may be used, e.g., as isolatedfrom bacteria or evolved/designed from a related lyase.

In some embodiments, the analytical process comprises detecting and/ormeasuring the one or more oligosaccharide present in the biologicalsample after it has been treated with one or more glycosaminoglycanlyase. In some embodiments, the one or more oligosaccharide detectedand/or measured is one or more disaccharide and/or one or moretrisaccharide. In certain embodiments, the one or more oligosaccharidesdetected and/or measured (e.g., one or more disaccharide and/or one ormore trisaccharide) are saturated at 4 and 5 carbons of the non-reducingend saccharide residue. In some embodiments, the non-reducing endresidue of the one or more oligosaccharides detected and/or measured(e.g., one or more disaccharide and/or one or more trisaccharide) arefree of carbon-carbon unsaturation. In certain embodiments, the one ormore oligosaccharides detected and/or measured (e.g., one or moredisaccharide and/or one or more trisaccharide) are free of carbon-carbonunsaturation. Biological samples suitable for analysis according to themethods and processes described herein include, by way of non-limitingexample, blood, serum, urine, hair, saliva, skin, tissue, plasma,cerebrospinal fluid (CSF), amniotic fluid, nipple aspirate, sputum,feces, synovial fluid, nails, or the like. In specific embodiments, thebiological samples suitable for analysis according to the methods andprocesses described herein include, by way of non-limiting example,urine, serum, plasma, or CSF. In certain embodiments, processes fordetecting glycosoaminoglycans in a sample comprise providing, from theindividual, a test biological sample that comprises glycosaminoglycans.In some embodiments, providing a test biological sample from anindividual includes obtaining the sample from the individual orobtaining the sample from another source (e.g., from a technician orinstitution that obtained the sample from the individual). In someembodiments, the biological sample is obtained from any suitable source,e.g., any tissue or cell (e.g., urine, serum, plasma, or CSF) of anindividual. In certain embodiments, the tissue and/or cell from whichthe GAGs are recovered is obtained from liver tissue or cells, braintissue or cells, kidney tissue or cells, or the like.

FIG. 1 illustrates the cleavage of the glycosaminoglycan (GAG) heparansulfate with a glycosaminoglycan lyase (heparinase II). As illustrated,in certain instances, internal cleavage of glycosaminoglycans withglycosaminoglycan lyases provides oligosaccharides with carbon-carbonunsaturation between the C4 and C5 carbons of the non-reducing end ofthe oligosaccharide produce (i.e., the newly created oligosaccharide).In some embodiments, the one or more oligosaccharide detected and/ormeasured according to a method described herein is one or moredisaccharide and/or one or more trisaccharide, each oligosaccharidebeing is comprised of two or three saccharide residues that formed theoriginal two or three saccharide residues of a glycosaminoglycan (GAG)prior to treatment with the one or more glycosaminoglycan (GAG) lyase.

In certain embodiments, analytical methods provided herein furthercomprise methods of purification. In certain embodiments, purificationmethods are performed prior to treating a biological sample with alyase, as described herein. In some embodiments, purification methodsare performed after treating a biological sample with a lyase, asdescribed herein. In certain embodiments, purification methods areutilized before and after treating a biological sample with a lyase, asdescribed herein. In some embodiments, purification methods includepurifying one or more glycosaminoglycan and/or one or moreoligosaccharide from other components (e.g., cells, cell parts, otherpolysaccharides, or the like) of the biological sample. In certainembodiments, purification methods include purifying one or moreglycosaminoglycan from other polysaccharides (e.g., other glycans, otherglycosaminoglycans, other sugars, or the like).

In certain instances the GAGs provided in a biological sample arepresent in lysosomes of cells. In some embodiments, any processdescribed herein includes lysing a biological sample to free the GAGsfrom the cells therein.

Diagnostics

Provided in some embodiments herein is a process for diagnosing theidentity and/or severity of abnormal glycosaminoglycan (or other glycan,e.g., glycolipid) accumulation in an individual, or a disorder thereof,the process comprising the step of: detecting the presence of and/ormeasuring the amount of a population of one or more oligosaccharidespresent in a transformed biological sample (e.g., urine, serum, plasma,or CSF). In certain embodiments, the process for diagnosing the identityand/or severity of abnormal glycosaminoglycan accumulation in anindividual is a process of diagnosing the individual as an individualsuffering from, homozygous for, or symptomatic for such a disorder. Inother embodiments, the process for diagnosing the identity and/orseverity of abnormal glycosaminoglycan accumulation in an individual isa process of diagnosing the individual as an individual suffering fromsuch a disorder as a carrier for, or heterozygous for, such a disorder.In some embodiments, individuals that are carriers for, or heterozygousfor, such a disorder has an elevated level of glycosaminoglycanaccumulation (e.g., when compared to a normal individual), but theelevated level is less than an individual diagnosed with having thedisorder. In certain embodiments, individuals that are carriers for, orheterozygous for, such a disorder has an elevated level ofglycosaminoglycan accumulation (e.g., when compared to a normalindividual), but are asymptomatic (including substantially asymptomatic)for a GAG accumulation disorder. Carriers and individuals having a GAGaccumulation disease are identified utilizing any appropriate procedure.For example, in certain embodiments, carriers or carrier specimens maybe identified as accumulating, e.g., 2-100 times more GAG than anon-carrier or wild type specimen. Similarly, in some exemplaryembodiments, individuals that are symptomatic or have a GAG accumulationdisease state accumulate more than 2 times more (e.g., 2-100×) GAG thana carrier. In some embodiments, diagnosis of one or more carrier parentis optionally utilized to make a progeny risk assessment (e.g.,likelihood of a child being a carrier for or having a disease state).

In some embodiments, provided herein is a process for diagnosingabnormal glycosaminoglycan accumulation in an individual, or a disorderthereof, the process comprising the step of: using an analyticalinstrument to detect the presence of and/or measure the amount of apopulation of one or more oligosaccharides present in a transformedbiological sample that has been prepared by treating a population ofglycosaminoglycans, in or isolated from a biological sample from theindividual, with at least one digesting glycosaminoglycan lyase totransform the glycosaminoglycans into the population of the one or moreoligosaccharide. In specific embodiments, the oligosaccharide(s)detected or measured is one or more C4-C5 non-reducing end saturatedoligosaccharide(s).

In some embodiments, provided herein is a process for diagnosing theidentity (or type, e.g., heparan sulfate, chondroitin sulfate, or anyother glycosaminoglycan) of abnormal glycosaminoglycan accumulation inan individual, or a disorder thereof, the process comprising the stepof: using an analytical instrument to detect the presence of and/ormeasure the amount of a population of one or more oligosaccharidespresent in a transformed biological sample that has been prepared bytreating a population of glycosaminoglycans, in or isolated from abiological sample from the individual, with at least one digestingglycosaminoglycan lyase to transform the glycosaminoglycans into thepopulation of the one or more oligosaccharide. In specific embodiments,the oligosaccharide(s) detected or measured is one or more C4-C5non-reducing end saturated oligosaccharide(s).

In some embodiments, provided herein is a process for diagnosing theseverity of abnormal glycosaminoglycan accumulation in an individual, ora disorder thereof, the process comprising the step of: using ananalytical instrument to detect the presence of and/or measure theamount of a population of one or more oligosaccharides present in atransformed biological sample that has been prepared by treating apopulation of glycosaminoglycans, in or isolated from a biologicalsample from the individual, with at least one digestingglycosaminoglycan lyase to transform the glycosaminoglycans into thepopulation of the one or more oligosaccharide. In specific embodiments,the oligosaccharide(s) detected or measured is one or more C4-C5non-reducing end saturated oligosaccharide(s).

In some embodiments, provided herein is a process for diagnosing anindividual as being a carrier of a gene that causes abnormalglycosaminoglycan accumulation in an individual, or a disorder thereof,the process comprising the step of: using an analytical instrument todetect the presence of and/or measure the amount of a population of oneor more oligosaccharides present in a transformed biological sample thathas been prepared by treating a population of glycosaminoglycans, in orisolated from a biological sample from the individual, with at least onedigesting glycosaminoglycan lyase to transform the glycosaminoglycansinto the population of the one or more oligosaccharide. In certaininstances, such a process involves determining the severity of abnormalglycosaminoglycan accumulation, wherein such accumulation is below acertain threshold (e.g., a predetermined level, a level whereby theindividual becomes symptomatic, or the like). In specific embodiments,the oligosaccharide(s) detected or measured is one or more C4-C5non-reducing end saturated oligosaccharide(s).

In some embodiments, provided herein is a process for diagnosingabnormal glycosaminoglycan accumulation in a human infant (e.g., anewborn) or fetus, or a disorder thereof, the process comprising thestep of: using an analytical instrument to detect the presence of and/ormeasure the amount of a population of one or more oligosaccharidespresent in a transformed biological sample that has been prepared bytreating a population of glycosaminoglycans, in or isolated from abiological sample from the individual, with at least one digestingglycosaminoglycan lyase to transform the glycosaminoglycans into thepopulation of the one or more oligosaccharide. In specific embodiments,the oligosaccharide(s) detected or measured is one or more C4-C5non-reducing end saturated oligosaccharide(s).

In further embodiments, any of the processes described herein furthercomprise the step of displaying or recording the presence of or ameasure of a population of one or more oligosaccharide. The display maybe on a computer screen or a paper print out. The recording may be onany computer readable disk (e.g., a hard drive, CD, DVD, portable memorydevice, such as a CF device or SD device, or the like), a sheet ofpaper, or the like.

In some embodiments, the transformed biological sample is prepared bytreating a population of glycosaminoglycans or other glycan (e.g.,glycolipid), the glycosaminoglycans or other glycan (e.g., glycolipid)being present in or isolated from a biological sample (e.g., urine,serum, plasma, or CSF) from an individual. Diagnostics, methods andcompositions of matter described herein when referring to a GAG ingeneral or a specific GAG, e.g., heparan sulfate, is understood tocontain disclosure for any suitable glycan (e.g., a glycolipid). Incertain embodiments, the glycosaminoglycans are treated with at leastone agent suitable for cleaving bonds between saccharide residues ofglycosaminoglycans. In some embodiments, a process described hereincomprises transforming a biological sample by treating a population ofglycosaminoglycans, the glycosaminoglycans being present in or isolatedfrom a biological sample from an individual. In certain embodiments, theglycosaminoglycans are treated with at least one agent suitable forcleaving bonds between saccharide residues of glycosaminoglycans. Inspecific embodiments, treating a biological sample that comprisesglycosaminoglycans with at least one agent suitable for cleaving bondsbetween saccharide residues of glycosaminoglycans comprises treating thebiological sample with one or more digesting glycosaminoglycan (GAG)lyase. In some embodiments, the one or more digesting glycosaminoglycanlyase is one or more heparin lyase, one or more chondroitinase, one ormore keratanase, one or more hyaluronidase, or a combination thereof. Incertain embodiments, treatment of the glycosaminoglycan with the lyaseprovides to transform the glycosaminoglycans into the population of theone or more oligosaccharide. In specific embodiments, the at least onedigesting glycosaminoglycan lyase is one or more heparin lyase.

In certain embodiments, the abnormal glycosaminoglycan accumulationcomprises abnormal heparan sulfate accumulation, abnormal chondroitinsulfate accumulation, abnormal keratan sulfate accumulation, abnormalhyaluronan accumulation, abnormal dermatan sulfate accumulation, or acombination thereof. In some embodiments, disorders associated withabnormal glycosaminoglycan accumulation include lysosomal storagediseases, such as, by way of non-limiting example, mucopolysaccharidosis(MPS) (e.g., MPS I, MPS II, MPS IIIA, MPS IIIB, MPS IIIC, MPS IIID, MPSIVA, MPS IVB, MPS VI, MPS VII, MPS IX, or the like). In someembodiments, the process of diagnosing the identity of or the severityof a disorder associated with the accumulation of glycosaminoglycans isa disorder associated with abnormal heparan sulfate accumulation. Inspecific embodiments, disorders associated with abnormal heparan sulfateaccumulation include, by way of non-limiting example, MPS I, MPS II, MPSIIIA, MPS IIIB, MPS IIIC, MPS IIID, MPS VII, or the like. In someembodiments, the process of diagnosing the identity of or the severityof a disorder associated with the accumulation of glycosaminoglycans isa disorder associated with abnormal dermatan sulfate accumulation (e.g.,in some instances, MPS I, MPS II, MPS VI, or the like). In certainembodiments, the process of diagnosing the identity of or the severityof a disorder associated with the accumulation of glycosaminoglycans isa disorder associated with abnormal chondroitin sulfate accumulation(e.g., in some instances, MPS VI, MPS VII, or the like). In someembodiments, the process of diagnosing the identity of or the severityof a disorder associated with the accumulation of glycosaminoglycans isa disorder associated with abnormal keratan sulfate accumulation (e.g.,in some instances, MPS IVA, MPS IVB, or the like). In certainembodiments, the process of diagnosing the identity of or the severityof a disorder associated with the accumulation of glycosaminoglycans isa disorder associated with abnormal hyaluronan accumulation (e.g., insome instances, MPS VII, MPS IX, or the like). In some embodiments,oligosaccharides provided by treating the glycosaminoglycan with asuitable glycosaminoglycan lyase are utilized in processes describedherein to diagnose the identity of and/or measure the severity of adisorder associated with the abnormal accumulation of the particularglycosaminoglycan. Specific oligosaccharides provided by treatingvarious glycosaminoglycans with glycosaminoglycan lyases are providedherein in the oligosaccharide section.

Moreover, in certain embodiments, the diagnostic methods describedherein (or other method described herein) are suitable for diagnosing(or measuring the efficacy of a treatment of) a disorder in anindividual involved with glycan (e.g., GAG) accumulation or any disorderinvolved with altered GAG synthesis and degradation (e.g., any disorderthat provides a unique GAG or population of GAGs that can be detected bya process described herein). In some embodiments, such a diseaseincludes Alzheimer's Disease, wherein GAGs are present in plaques, and abiological sample is taken from the plaque and analyzed according to aprocess described herein. In other embodiments, such a disease includescancer.

In some embodiments, specific oligosaccharides are detected and/ormeasured according to methods and/or processes described herein todiagnose the identity and/or severity of a specific disorder associatedwith glycosaminoglycan accumulation. In some embodiments, sucholigosaccharides are described herein. In specific embodiments, aprocess for diagnosing the identity or severity of a disorder associatedwith the accumulation of glycosaminoglycans provided herein comprisesdetecting and/or measuring one or more oligosaccharide set forth inFormulas I-XX or any other oligosaccharide described in the figures. Incertain embodiments, the one or more oligosaccharides detected and/ormeasured are free of carbon-carbon unsaturation. In some embodiments,the one or more oligosaccharides detected and/or measured are free of C4and C5 carbon unsaturation on the saccharide residue at the non-reducingend of the oligosaccharide. In some embodiments, the oligosaccharide ofany of Formulas I-XX is a disaccharide or trisaccharide comprised of twoor three saccharide residues that formed the original two or threesaccharide residues of a glycosaminoglycan (GAG) prior to treatment withthe one or more glycosaminoglycan (GAG) lyase. In certain instances, theamount of disaccharide or trisaccharide of any of Formulas I-XX free ofnon-reducing end carbon-carbon (e.g., C4/C5) is representative of theamount of accumulated glycosaminoglycans comprising the samedisaccharide or trisaccharide as residue thereof, at its non-reducingend.

In certain instances, a diagnostic method described herein is useful foranalyzing the various different classes of MPS. In some instances, theGAG accumulation provides a unique population of GAGs depending on thespecific MPS class. In specific instance, the unique population of GAGscan be identified as being correlated with a specific MPS class bydetecting and/or measuring oligosaccharides in a sample taken from anindividual diagnosed with or suspected of having an MPS disorder, theoligosaccharides being free of C4 and C5 carbon unsaturation on thesaccharide residue at the non-reducing end of the oligosaccharide. Incertain instances, the oligosaccharides are digested with a suitableenzyme, such as a lyase (e.g., a bacterial lyase or heparin lyase) priorto detection/measurement and the resulting oligosaccharide (shorter incertain instances than the sample oligosaccharide, such as di- ortri-saccharides) are detected/measured. In certain instances, thedegradation enzymes (e.g., heparin lyase) work by an eliminase mechanismwhich introduces an unsaturated bond on the newly generated non-reducingend; whereas preexisting non-reducing ends retain their full mass (e.g.,these non-reducing ends are free of C4 and C5 carbon unsaturation). Thusin certain embodiments, the digested oligosaccharides comprisingnon-reducing ends that are free of C4 and C5 carbon unsaturation arerepresentative of the total number of oligosaccharides present in theoriginal sample composition. In certain instances, the mechanism ofdigesting (e.g., with a heparin lyase) effectively tags the preexistingends to allow for their identification by their unique mass (e.g., being18 Daltons larger than the other oligosaccharides provided by internaloligosaccharide residues). In some instances, identification of thesepreexisting non-reducing ends are excellent biomarkers because, e.g., incertain instances (1) they are homogenous within an MPS class (e.g., incertain instances for MPS II, they all end in 2-O sulfated uronic acid);(2) there are many more non-reducing ends in GAGs from individualssuffering from MPS than in non-MPS individuals); and/or (3) in non-MPSindividuals the non-reducing end saccharide residues are heterogeneous(see, e.g., FIG. 2). FIG. 3A illustrates the large number ofoligosaccharide residues found within GAGs and FIGS. 3B and 3C summarizethe predicted non-reducing end heparan sulfate oligosaccharide residues(e.g., the biomarkers) for 7 MPS classes. In some embodiments, any oneor more of these oligosaccharides are detected and/or measured in amethod of diagnosing an individual suffering from the specific MPS classdescribed. In some embodiments, additional biomarkers for these MPSclasses that originate from chondroitin and dermatan can also beanalyzed using the same method. The same approach (using chondroitinase,keratanase, and hyaluronidases) is provided for in certain embodimentsfor other MPS classes that accumulate CS, DS, KS, and/or HA.

In certain embodiments, a process for diagnosing the identity orseverity of a disorder associated with the accumulation ofglycosaminoglycans provided herein comprises detecting and/or measuringone or more oligosaccharide set forth in Formulas XXI-XXIX or in any ofthe figures described herein. In certain embodiments, the one or moreoligosaccharides detected and/or measured comprise at least one point ofcarbon-carbon unsaturation. In some embodiments, the one or moreoligosaccharides detected and/or measured comprise C4 and C5 carbonunsaturation on the saccharide residue at the non-reducing end of theoligosaccharide.

In certain embodiments, processes described herein, including diagnosticprocesses, include preparing a transformed biological sample bypurifying a population of oligosaccharides in a biological sample thathas been treated with the at least one glycosaminoglycan lyase (e.g.,one or more heparin lyase), the transformed biological sample comprisingthe isolated population of oligosaccharides. In some embodiments,glycosaminoglycans of the biological sample from an individual arepurified prior to treatment with the one or more glycosaminoglycanlyase.

In some embodiments, a diagnostic (including identity or severitydiagnostic) process provided herein comprises comparing a detection ormeasurement according to the process to a control reading. In someembodiments, the comparison to a control comprises comparing the amountof the population of one or more oligosaccharide present in thetransformed biological sample to an amount of a population of the one ormore oligosaccharide present in a control biological sample that hasbeen treated in a manner substantially similar to the transformedbiological sample. In specific embodiments, the control biologicalsample was provided from an individual that does not have a disorderassociated with abnormal glycosaminoglycan accumulation (e.g.,mucopolysaccharidosis (MPS)). In specific embodiments, the controlbiological sample was provided from an individual that has a disorderassociated with abnormal glycosaminoglycan accumulation (e.g.,mucopolysaccharidosis (MPS)). In more specific embodiments, the controlis from an individual with an abnormal glycosaminoglycan accumulationselected from, by way of non-limiting example, MPS I, MPS II, MPS IIIA,MPS IIIB, MPS IIIC, MPS IIID, MPS IVA, MPS IVB, MPS VI, MPS VII, MPS IX,and a combination thereof (e.g., MPS I, MPS II, MPS IIIA, MPS IIIB, MPSIIIC, MPS IIID, MPS VII, and a combination thereof).

In some embodiments, detecting the presence of or measuring the amountof a population of one or more oligosaccharide present in a transformedbiological sample according to a process described herein comprises:

-   -   a. isolating a subpopulation of one or more oligosaccharides in        the transformed biological sample (e.g., a transformed urine,        serum, plasma, or CSF sample); and    -   b. detecting the presence of and/or measuring the amount of one        or more oligosaccharides present in the subpopulation.

Isolation of the subpopulation of one or more oligosaccharides in thetransformed biological sample is achieved in any suitable manner, e.g.,using a purification process described herein (e.g., chromatography,electrophoresis, filtration, centrifugation, etc.). Similarly, accordingto any process described herein, the detection of and/or measuring thepresence of one or more oligosaccharide is achieved utilizing anysuitable process, including those detection processes set forth herein(e.g., spectrometry, UV-Visible spectrometry, IR spectrometry, NMRspectrometry, mass spectrometry, or the like). In specific instances,prior to detecting and/or measuring the oligosaccharide present, anyprocess described herein further comprises tagging the reducing end of arepresentative portion of the one or more oligosaccharides in thetransformed biological sample with any suitable detectable label (e.g.,a mass label, a radio label, a fluorescent label, a chromophore label,affinity label, etc.).

In certain embodiments, the detection of the presence and/or measure ofthe amount of oligosaccharide is performed utilizing an analyticalinstrument. In specific embodiments, the analytical device comprises aspectrometer that detects and/or measures the amount of a detectablelabel. In certain embodiments, the detection and/or measurement ofamounts of a detectable label serves as a proxy to the presence oramounts of GAGs present. In more specific embodiments, the spectrometerincludes, by way of non-limiting example, one or more of a massspectrometer, a nuclear magnetic resonance spectrometer, a UV-Visspectrometer, an IR spectrometer, a fluorimeter, a phosphorimeter, aradiation spectrometer, or the like. In certain embodiments, theanalytical device comprises a purification device coupled to a detectoror a measuring device (e.g., a HPLC system coupled to a UV-Visspectrometer). In certain embodiments, an analytical device is a liquidchromatography mass spectrometer (LC-MS) that detects and/or measuresthe mass of an oligosaccharide.

In some embodiments, the presence detected and/or the measure of thepopulation of the oligosaccharide is displayed or recorded. In someembodiments, the process comprises displaying or recording the resultsof the characterization. In certain embodiments, the results aredisplayed on a display monitor (e.g., a computer monitor, television,PDA, or the like), or print out. In some embodiments, the results arerecorded on an electronic medium (e.g., a hard disk drive, magneticstorage drive, optical storage drive or the like; a disk such as afloppy disk, CD, DVD, BLU-ray or the like; a flash memory drive;removable drive or the like).

In certain embodiments, the individual is a mammal, e.g., a human. Insome embodiments, the human is a newborn. In certain embodiments, thehuman is an embryo in utero. In some embodiments, the human has beendiagnosed with a lysosomal storage disease. In some embodiments, thehuman is suspected of suffering from a lysosomal storage disease.

Analytical Samples

Provided in certain embodiments herein are compositions comprising anyone or more oligosaccharides provided herein. In some embodiments, thecomposition provided herein is an analytical sample, suitable analysisin any analytical device, e.g., one provided herein (such as, by way ofnon-limiting example, high performance liquid chromatography, massspectrometry, gas chromatography, or the like).

In certain embodiments, a composition provided herein comprises at leastone disaccharide or trisaccharide from a transformed biological samplefrom an individual with a disorder associated with abnormalglycosaminoglycan accumulation. In specific embodiments, the transformedbiological sample was prepared by treating a biological samplecomprising glycosaminoglycans with one or more digestingglycosaminoglycan lyase.

In some embodiments, an analytical sample provided herein comprises oneor more oligosaccharide of any of Formulas Ito XX, any one of FormulasXXI to XXIX, or any of Formulas I to XXIX. In certain embodiments, ananalytical sample provided herein comprises one or more oligosaccharideof any of Formulas Ito XX, any one of Formulas XXI to XXIX, or any ofFormulas I to XXIX, wherein the one or more oligosaccharides furthercomprise a detectable label attached (e.g., covalently and/ornon-covalently) to the reducing end of the one or more oligosaccharide.

In some embodiments, provided herein is a composition comprisingisolated glycans, wherein the glycans were isolated from a biologicalsample, and one or more glycan degradation enzyme. In certainembodiments, the composition further comprises one or more biomarkergenerated according to any method described herein (e.g., wherein thebiomarker is a non-reducing saturated oligosaccharide). In certainembodiments, provided herein is an oligosaccharide described herein(e.g., a labeled or non-labeled non-reducing saturated oligosaccharide)and an analytical instrument or chromatographic resin.

Oligosaccharides

In certain embodiments, methods and processes described herein areutilized to detect and/or measure one or more biomarker. In specificembodiments, such biomarkers comprise one or more oligosaccharides(e.g., disaccharide(s) and/or trisaccharide(s)). In certain embodiments,the one or more oligosaccharides comprise any one or more of theoligosaccharides described herein.

As used herein, IdoA and

are iduronic acid (e.g., α-L-iduronic acid) saccharide residues. As usedherein, GlcAand are glucuronic acid (e.g., (β-L-glucuronic acid) saccharideresidues. As used herein,is either an iduronic acid (e.g., α-L-iduronic acid) saccharide residueor a glucuronic acid (e.g., (β-L-glucuronic acid) saccharide residue. Asused herein, GlcN andare glucosamine (e.g., 2-deoxy-2-amino-(β-D-glucopyranosyl) saccharideresidues. As used herein, GlcN(Ac)₁ andare a glucosamine (e.g., 2-deoxy-2-amino-(β-D-glucopyranosyl) saccharideresidue wherein the 2-amino group is acetylated. As used herein, Gal andis a galactose saccharide residue. As used herein GalNAc andrepresents an N-acetylgalactosamine residue. As used hereinandboth represent N-sulfated (i.e., N-substituted with SO₃R as describedherein) glucosamine (e.g., 2-deoxy-2-amino-(β-D-glucopyranosyl)saccharide residue. In various specific instances, iduronic acid,glucuronic acid, glucosamine, and/or galactose saccharide residues aresaturated at 4 and 5 carbons of the non-reducing end saccharide residue,or are free of carbon-carbon unsaturation. In other instances, any oneor more of the saccharide residues is unsaturated, e.g., at the 4 and 5carbon positions of the saccharide residue at the non-reducing end of anoligosaccharide provided herein. The symbolic nomenclature used hereinfollows the “Symbol and Text Nomenclature for Representation of GlycanStructure” as promulgated by the Nomenclature Committee for theConsortium for Functional Glycomics, as amended on October 2007.Recitation of an NS (e.g., above of below any of the aforementionedstructures) indicates that the amino group thereof is substituted with(SO₃R). If the NS is associated with GlcN(Ac)_(m) or above or below

, the residue is GlcN(SO₃R), wherein the amino group bears the (SO₃R).Recitation of a 2S (e.g., above or below any of the aforementionedstructures) indicates that the hydroxyl group at the two carbon positionof the indicated saccharide residue is substituted with (SO₃R).Recitation of a 3S (e.g., above of below any of the aforementionedstructures) indicates that the hydroxyl group at the three carbonposition of the indicated saccharide residue is substituted with (SO₃R).Recitation of a 4S (e.g., above of below any of the aforementionedstructures) indicates that the hydroxyl group at the four carbonposition of the indicated saccharide residue is substituted with (SO₃R).Recitation of a 6S (e.g., above of below any of the aforementionedstructures) indicates that the hydroxyl group at the six carbon positionof the indicated saccharide residue is substituted with (SO₃R).

In specific embodiments, methods and processes described herein areutilized to detect and/or measure an oligosaccharide (disaccharide)having the formula:

[IdoA-GlcN(Ac)_(m)](SO₃R)_(n)   Formula I

In certain embodiments, oligosaccharides described herein, e.g., thosedetected and/or measured according to the methods and/or processesdescribed herein, are disaccharides of Formula I, wherein m is 0-1, andn is 0-3. As used herein, each R is independently H or a negativecharge. The disaccharide is optionally sulfated with n SO₃R groups inany suitable location. In more specific embodiments, a disaccharide ofFormula I has a structure of IdoA-GlcNAc, IdoA-GlcNS, IdoA-GlcNS6S,IdoA2S-GlcNAc, IdoA2S-GlcNS, IdoA2S-GlcNAc6S, IdoA2S-GlcNS6S or as setforth in any of Formulas I-A to I-G, as set forth in FIG. 4. In certaininstances, compounds of Formulas I-A to I-G are provided by treating theglycosaminoglycan heparan sulfate with a suitable glycosaminoglycanlyase.

In some embodiments, the detection and/or measurement of any one or moredisaccharide of Formulas I-A to I-C is utilized in a process fordiagnosing MPS I. In certain embodiments, the detection and/ormeasurement of any one or more disaccharide of Formulas I-D to I-G isutilized in a process for diagnosing MPS II.

In certain embodiments, methods and processes described herein areutilized to detect and/or measure an oligosaccharide (trisaccharide)having the formula:

[GlcN(Ac)_(m)-(IdoA/GlcA)-GlcN(Ac)_(n)](SO₃R)_(p)   Formula II

In certain embodiments, oligosaccharides described herein, e.g., thosedetected and/or measured according to the methods and/or processesdescribed herein, are trisaccharides of Formula II, wherein IdoA/GlcA iseither IdoA or GlcA, m is 0-1, n is 0-1, and p is 0-5. The trisaccharideis optionally sulfated with p SO₃R groups in any suitable location.

In some specific embodiments, a compound of Formula II has a structureas set forth in Formula III:

[GlcNS-IdoA-GlcN(Ac)_(n)](SO₃R)_(p)   Formula III

In certain embodiments, oligosaccharides described herein, e.g., thosedetected and/or measured according to the methods and/or processesdescribed herein, are trisaccharides of Formula III, wherein n is 0-1,and p is 0-3. The trisaccharide is optionally sulfated with p SO₃Rgroups in any suitable location. In more specific embodiments, atrisaccharide of Formula III has a structure of GlcNS-IdoA-GlcNAc,GlcNS-IdoA2S-GlcNAc, GlcNS-IdoA-GlcNS, GlcNS-IdoA2S-GlcNS,GlcNS-IdoA-GlcNAc6S, GlcNS-IdoA2S-GlcNAc6S, GlcNS-IdoA-GlcNS6S,GlcNS-IdoA2S-GlcNS6S, or as set forth in any of the trisaccharides ofFIG. 5. In certain instances, compounds of Formulas III-A to III-H areprovided by treating the glycosaminoglycan heparan sulfate with asuitable glycosaminoglycan lyase.

In certain specific embodiments, a compound of Formula II has astructure as set forth in Formula IV:

[GlcNS-GlcA-GlcN(Ac)_(n)](SO₃R)_(p)   Formula IV

In certain embodiments, oligosaccharides described herein, e.g., thosedetected and/or measured according to the methods and/or processesdescribed herein, are trisaccharides of Formula IV, wherein n is 0-1,and p is 0-2. The trisaccharide is optionally sulfated with p SO₃Rgroups in any suitable location. In more specific embodiments, atrisaccharide of Formula IV has a structure of GlcNS-GlcA-GlcNAc,GlcNS-GlcA-GlcNS, GlcNS-GlcA-GlcNAc6S, GlcNS-GlcA-GlcNS6S or as setforth in any of the trisaccharides of FIG. 6. In certain instances,compounds of Formulas IV-A to IV-D are provided by treating theglycosaminoglycan heparan sulfate with a suitable glycosaminoglycanlyase.

In some embodiments, the detection and/or measurement of any one or moretrisaccharide of Formulas III and/or IV is utilized in a process fordiagnosing MPS IIIA.

In some specific embodiments, a compound of Formula II has a structureas set forth in Formula V:

[GlcNAc-IdoA-GlcN(Ac)_(n)](SO₃R)_(p)   Formula V

In certain embodiments, oligosaccharides described herein, e.g., thosedetected and/or measured according to the methods and/or processesdescribed herein, are trisaccharides of Formula V, wherein n is 0-1, andp is 0-3. The trisaccharide is optionally sulfated with p SO₃R groups inany suitable location. In more specific embodiments, a trisaccharide ofFormula V has a structure of GlcNAc-IdoA-GlcNAc, GlcNAc-IdoA2S-GlcNAc,GlcNAc-IdoA-GlcNS, GlcNAc-IdoA2S-GlcNS, GlcNAc-IdoA-GlcNAc6S,GlcNAc-IdoA2S-GlcNAc6S, GlcNAc-IdoA-GlcNS6S, GlcNAc-IdoA2S-GlcNS6, or asset forth in any of the trisaccharides of FIG. 7. In certain instances,compounds of Formulas V-A to V-H are provided by treating theglycosaminoglycan heparan sulfate with a suitable glycosaminoglycanlyase.

In certain specific embodiments, a compound of Formula II has astructure as set forth in Formula VI:

[GlcNAc-GlcA-GlcN(Ac)_(n)](SO₃R)_(p)   Formula VI

In certain embodiments, oligosaccharides described herein, e.g., thosedetected and/or measured according to the methods and/or processesdescribed herein, are trisaccharides of Formula VI, wherein n is 0-1,and p is 0-2. The trisaccharide is optionally sulfated with p SO₃Rgroups in any suitable location. In more specific embodiments, atrisaccharide of Formula VI has a structure of GlcNAc-GlcA-GlcNAc,GlcNAc-GlcA-GlcNS, GlcNAc-GlcA-GlcNAc6S, GlcNAc-GlcA-GlcNS6S or as setforth in any of the trisaccharides of FIG. 8. In certain instances,compounds of Formulas VI-A to VI-D are provided by treating theglycosaminoglycan heparan sulfate with a suitable glycosaminoglycanlyase.

In some embodiments, the detection and/or measurement of any one or moretrisaccharide of Formulas V and/or VI is utilized in a process fordiagnosing MPS IIIB.

In some specific embodiments, a compound of Formula II has a structureas set forth in Formula VII:

[GlcN-Ido-GlcN(Ac)_(n)](SO₃R)_(p)   Formula VII

In certain embodiments, oligosaccharides described herein, e.g., thosedetected and/or measured according to the methods and/or processesdescribed herein, are trisaccharides of Formula VII, wherein n is 0-1,and p is 0-4. The trisaccharide is optionally sulfated with p SO₃Rgroups in any suitable location. In more specific embodiments, atrisaccharide of Formula VII has a structure of GlcN-IdoA-GlcNAc,GlcN-IdoA2S-GlcNAc, GlcN-IdoA-GlcNS, GlcN-IdoA2S-GlcNS,GlcN-IdoA-GlcNAc6S, GlcN-IdoA2S-GlcNAc6S, GlcN-IdoA-GlcNS6S,GlcN-IdoA2S-GlcNS6S, or as set forth in any of the trisaccharides ofFIG. 9. In certain instances, compounds of Formulas VII-A to VII-H areprovided by treating the glycosaminoglycan heparan sulfate with asuitable glycosaminoglycan lyase.

In certain specific embodiments, a compound of Formula II has astructure as set forth in Formula VIII:

[GlcN-GlcA-GlcN(Ac)_(n)](SO₃R)_(p)   Formula VIII

In certain embodiments, oligosaccharides described herein, e.g., thosedetected and/or measured according to the methods and/or processesdescribed herein, are trisaccharides of Formula VIII, wherein n is 0-1,and p is 0-4. The trisaccharide is optionally sulfated with p SO₃Rgroups in any suitable location. In more specific embodiments, atrisaccharide of Formula VIII has a structure of GlcN-GlcA-GlcNAc,GlcN-GlcA-GlcNS, GlcN-GlcA-GlcNAc6S, GlcN-GlcA-GlcNS6S, or as set forthin any of the trisaccharides of FIG. 10. In certain instances, compoundsof Formulas VIII-A to VIII-D are provided by treating theglycosaminoglycan heparan sulfate with a suitable glycosaminoglycanlyase.

In some embodiments, the detection and/or measurement of any one or moretrisaccharide of Formulas VII and/or VIII is utilized in a process fordiagnosing MPS IIIC.

In some specific embodiments, a compound of Formula II has a structureas set forth in Formula IX:

[GlcNAc6S-Ido-GlcN(Ac)_(n)](SO₃R)_(p)   Formula IX

In certain embodiments, oligosaccharides described herein, e.g., thosedetected and/or measured according to the methods and/or processesdescribed herein, are trisaccharides of Formula IX, wherein n is 0-1,and p is 0-3. The trisaccharide is optionally sulfated with p SO₃Rgroups in any suitable location. In more specific embodiments, atrisaccharide of Formula VII has a structure of GlcNAc6S-IdoA-GlcNAc,GlcNAc6S-IdoA2S-GlcNAc, GlcNAc6S-IdoA-GlcNS, GlcNAc6S-IdoA2S-GlcNS,GlcNAc6S-IdoA-GlcNAc6S, GlcNAc6S-IdoA2S-GlcNAc6S, GlcNAc6S-IdoA-GlcNS6S,GlcNAc6S-IdoA2S-GlcNS6S, or as set forth in any of the trisaccharides ofFIG. 11. In certain instances, compounds of Formulas IX-A to IX-H areprovided by treating the glycosaminoglycan heparan sulfate with asuitable glycosaminoglycan lyase.

In certain specific embodiments, a compound of Formula II has astructure as set forth in Formula X:

[GlcNAc6S-GlcA-GlcN(Ac)_(n)](SO₃R)_(p)   Formula X

In certain embodiments, oligosaccharides described herein, e.g., thosedetected and/or measured according to the methods and/or processesdescribed herein, are trisaccharides of Formula X, wherein n is 0-1, andp is 0-2. The trisaccharide is optionally sulfated with p SO₃R groups inany suitable location. In more specific embodiments, a trisaccharide ofFormula X has a structure of GlcNAc6S-GlcA-GlcNAc, GlcNAc6S-GlcA-GlcNS,GlcNAc6S-GlcA-GlcNAc6S, GlcNAc6S-GlcA-GlcNS6S, or as set forth in any ofthe trisaccharides of FIG. 12. In certain instances, compounds of

Formulas X-A to X-D are provided by treating the glycosaminoglycanheparan sulfate with a suitable glycosaminoglycan lyase.

In some embodiments, the detection and/or measurement of any one or moretrisaccharide of Formulas IX and/or X is utilized in a process fordiagnosing MPS IIID.

In certain embodiments, methods and processes described herein areutilized to detect and/or measure an oligosaccharide (disaccharide)having the formula:

[GlcA-GlcN(Ac)_(m)](SO₃R)_(n)   Formula XI

In certain embodiments, oligosaccharides described herein, e.g., thosedetected and/or measured according to the methods and/or processesdescribed herein, are disaccharides of Formula XI, wherein m is 0-1, andn is 0-2. The disaccharide is optionally sulfated with n SO₃R groups inany suitable location. In more specific embodiments, a disaccharide ofFormula XI has a structure of GlcA-GlcNAc, GlcA-GlcNAc 6S, GlcA-GlcNS,GlcA-GlcNS6S, or as set forth in any of the disaccharides of FIG. 13. Incertain instances, compounds of Formulas XI-A to XI-D are provided bytreating the glycosaminoglycan heparan sulfate with a suitableglycosaminoglycan lyase.

In some embodiments, the detection and/or measurement of any one or moredisaccharide of Formula XI is utilized in a process for diagnosing MPSVII.

In certain embodiments, methods and processes described herein areutilized to detect and/or measure an oligosaccharide (disaccharide)having the formula:

[IdoA-GalNAc](SO₃R)_(n)   Formula XII

In certain embodiments, oligosaccharides described herein, e.g., thosedetected and/or measured according to the methods and/or processesdescribed herein, are disaccharides of Formula XII, wherein n is 0-2.The disaccharide is optionally sulfated with n SO₃R groups in anysuitable location. In more specific embodiments, a disaccharide ofFormula XII has a structure of IdoA-GalNAc, IdoA-GalNAc6S,IdoA-GalNAc4S, IdoA-GalNAc4S6S, or as set forth in any of thedisaccharides of FIG. 14. In certain instances, compounds of FormulasXII-A to XII-D are provided by treating the glycosaminoglycan dermatansulfate with a suitable glycosaminoglycan lyase.

In some embodiments, the detection and/or measurement of any one or moredisaccharide of Formula XII is utilized in a process for diagnosing MPSI. In certain embodiments, the detection and/or measurement of any oneor more disaccharide of Formula XII and one or more disaccharide ofFormulas I-A to I-C is utilized in a process for diagnosing MPS I.

In certain embodiments, methods and processes described herein areutilized to detect and/or measure an oligosaccharide (disaccharide)having the formula:

[IdoA2S-GalNAc](SO₃R)_(n)   Formula XIII

In certain embodiments, oligosaccharides described herein, e.g., thosedetected and/or measured according to the methods and/or processesdescribed herein, are disaccharides of Formula XIII, wherein n is 0-2.The disaccharide is optionally sulfated with n SO₃R groups in anysuitable location. In more specific embodiments, a disaccharide ofFormula XIII has a structure of IdoA2S-GalNAc, IdoA2S-GalNAc6S,IdoA2S-GalNAc4S, IdoA2S-GalNAc4S6S, or as set forth in any of thedisaccharides of FIG. 15. In certain instances, compounds of FormulasXIII-A to XIII-D are provided by treating the glycosaminoglycan dermatansulfate with a suitable glycosaminoglycan lyase.

In some embodiments, the detection and/or measurement of any one or moredisaccharide of Formula XIII is utilized in a process for diagnosing MPSII. In certain embodiments, the detection and/or measurement of any oneor more disaccharide of Formula XIII and one or more disaccharide ofFormulas I-D to I-G is utilized in a process for diagnosing MPS II.

In certain embodiments, methods and processes described herein areutilized to detect and/or measure an oligosaccharide (trisaccharide)having the formula:

[GalNAc4S-IdoA-GalNAc](SO₃R)_(n) Formula XIV

In certain embodiments, oligosaccharides described herein, e.g., thosedetected and/or measured according to the methods and/or processesdescribed herein, are trisaccharides of Formula XIV, wherein n is 0-4.The trisaccharide is optionally sulfated with n SO₃R groups in anysuitable location. In more specific embodiments, a disaccharide ofFormula XIV has a structure of GalNAc4S(±6S)-IdoA(±2S)-GalNAc(±4S)(±6S),or as set forth in any of the disaccharides of FIG. 16, wherein ±indicates that the indicated sulfation is independently present orabsent. In certain instances, compounds of Formulas XIV-A to XIV-P areprovided by treating the glycosaminoglycan dermatan sulfate with asuitable glycosaminoglycan lyase.

In some embodiments, the detection and/or measurement of any one or moretrisaccharide of Formula XIV is utilized in a process for diagnosing MPSVI.

In certain embodiments, methods and processes described herein areutilized to detect and/or measure an oligosaccharide (trisaccharide)having the formula:

[GalNAc4S-GlcA-GalNAc](SO₃R)_(n)   Formula XV

In certain embodiments, oligosaccharides described herein, e.g., thosedetected and/or measured according to the methods and/or processesdescribed herein, are trisaccharides of Formula XV, wherein n is 0-3.The trisaccharide is optionally sulfated with n SO₃R groups in anysuitable location. In more specific embodiments, a disaccharide ofFormula XV has a structure of GalNAc4S(±6S)-GlcA(±2S)-GlcNAc(±4S)(±6S),or as set forth in any of the trisaccharides of FIG. 17, wherein ±indicates that the indicated sulfation is independently present orabsent. In certain instances, compounds of Formulas XV-A to XV-P areprovided by treating the glycosaminoglycan chondroitin sulfate with asuitable glycosaminoglycan lyase.

In some embodiments, the detection and/or measurement of any one or moretrisaccharide of Formula XV is utilized in a process for diagnosing MPSVI. In certain embodiments, the detection and/or measurement of any oneor more trisaccharide of Formula XIV and one or more trisaccharide ofFormula XV is utilized in a process for diagnosing MPS VI.

In certain embodiments, methods and processes described herein areutilized to detect and/or measure an oligosaccharide (trisaccharide)having the formula:

GlcA-GlcNAc-GlcA   Formula XVI

In certain embodiments, oligosaccharides described herein, e.g., thosedetected and/or measured according to the methods and/or processesdescribed herein, are trisaccharides of Formula XVI. In certaininstances, compounds of Formulas XVI are provided by treating theglycosaminoglycan hyaluronan with a suitable glycosaminoglycan lyase. Insome embodiments, the detection and/or measurement of any one or moretrisaccharide of Formula XVI is utilized in a process for diagnosing MPSIIIB. In certain embodiments, the detection and/or measurement of anyone or more trisaccharide of Formula XVI and one or more trisaccharideof either of Formulas V and/or VI is utilized in a process fordiagnosing MPS BIB.

In certain embodiments, methods and processes described herein areutilized to detect and/or measure an oligosaccharide (disaccharide)having the formula:

GlcA-GlcNAc   Formula XVII

In certain embodiments, oligosaccharides described herein, e.g., thosedetected and/or measured according to the methods and/or processesdescribed herein, are disaccharides of Formula XVII. In certaininstances, compounds of Formulas XVII are provided by treating theglycosaminoglycan hyaluronan with a suitable glycosaminoglycan lyase. Insome embodiments, the detection and/or measurement of any one or moredisaccharide of Formula XVII is utilized in a process for diagnosing MPSVII or MPS IX. In certain embodiments, the detection and/or measurementof a disaccharide of Formula XVII and one or more disaccharide ofFormula XI is utilized in a process for diagnosing MPS VII. In someembodiments, the detection and/or measurement of a disaccharide ofFormula XVII and the absence of one or more disaccharide of Formula XIis utilized in a process for diagnosing MPS IX.

In certain embodiments, methods and processes described herein areutilized to detect and/or measure an oligosaccharide (trisaccharide)having the formula:

[GlcNAc6S-Gal-GlcNAc](SO₃R)_(n) Formula XVIII

In certain embodiments, oligosaccharides described herein, e.g., thosedetected and/or measured according to the methods and/or processesdescribed herein, are trisaccharides of Formula XVIII, and wherein n is0-2. The trisaccharide is optionally sulfated with n SO₃R groups in anysuitable location. In more specific embodiments, a disaccharide ofFormula XVIII has a structure of GlcNAc6S-Gal-GlcNAc,GlcNAc6S-Gal6S-GlcNAc, GlcNAc6S-Gal-GlcNAc6S, GlcNAc6S-Gal6S-GlcNAc6S,or as set forth in any of the trisaccharides of FIG. 18. In certaininstances, compounds of Formulas XVIII-A to XVIII-D are provided bytreating the glycosaminoglycan keratan sulfate with a suitableglycosaminoglycan lyase.

In some embodiments, the detection and/or measurement of any one or moretrisaccharide of Formula XVIII is utilized in a process for diagnosingMPS IIID. In certain embodiments, the detection and/or measurement ofany one or more trisaccharide of Formula XVIII and one or moretrisaccharide of Formulas IX and/or X is utilized in a process fordiagnosing MPS IIID.

In certain embodiments, methods and processes described herein areutilized to detect and/or measure an oligosaccharide (disaccharide)having the formula:

[Gal6S-GlcNAc-(Gal)_(m)](SO₃R)_(n) Formula XIX

In certain embodiments, oligosaccharides described herein, e.g., thosedetected and/or measured according to the methods and/or processesdescribed herein, are disaccharides of Formula XIX, wherein n is 0-2 andm is 0-1. The disaccharide is optionally sulfated with n SO₃R groups inany suitable location. In more specific embodiments, a disaccharide ofFormula XIX has a structure of Gal6S-GlcNAc, Gal6S-G1cNAc6S, or as setforth in any of the disaccharides of FIG. 19. In certain instances,compounds of Formulas XIX-A to XIX-B are provided by treating theglycosaminoglycan keratan sulfate with a suitable glycosaminoglycanlyase.

In some embodiments, the detection and/or measurement of any one or moredisaccharide of Formula XIX is utilized in a process for diagnosing MPSIVA.

In certain embodiments, methods and processes described herein areutilized to detect and/or measure an oligosaccharide (disaccharide)having the formula:

[Gal-G1cNAc](SO₃R)_(n)   Formula XX

In certain embodiments, oligosaccharides described herein, e.g., thosedetected and/or measured according to the methods and/or processesdescribed herein, are disaccharides of Formula XX, wherein n is 0-1. Thedisaccharide is optionally sulfated with n SO₃R groups in any suitablelocation. In more specific embodiments, a disaccharide of Formula XX hasa structure of Gal-GlcNAc, Gal-GlcNAc6S, or as set forth in any of thedisaccharides of FIG. 20. In certain instances, compounds of FormulasXX-A to XX-B are provided by treating the glycosaminoglycan keratansulfate with a suitable glycosaminoglycan lyase.

In some embodiments, the detection and/or measurement of any one or moredisaccharide of Formula XX is utilized in a process for diagnosing MPSIVB.

In certain embodiments, any analytical method or diagnostic processdescribed herein comprises detecting and/or measuring any one or more ofthe oligosaccharides of any of Formulas I-XX, wherein the non-reducingend saccharide residue is saturated at 4 and 5 carbons of thenon-reducing end saccharide residue, the non-reducing end saccharideresidue is free of carbon-carbon unsaturation, or the oligosaccharide isfree of carbon-carbon unsaturation. In certain instances, thenon-reducing end saccharide residue is on the left end of theoligosaccharides disclosed herein. FIG. 1 illustrates the cleavage ofthe glycosaminoglycan (GAG) heparan sulfate with a glycosaminoglycanlyase (heparinase II). As illustrated, in certain instances, internalcleavage of glycosaminoglycans with glycosaminoglycan lyases providesoligosaccharides with carbon-carbon unsaturation between the C4 and C5carbons of the non-reducing end of the oligosaccharide produce (i.e.,the newly created oligosaccharide). In some embodiments, theoligosaccharide of any of Formulas I-XX is a disaccharide ortrisaccharide comprised of two or three saccharide residues that formedthe original two or three saccharide residues of a glycosaminoglycan(GAG) prior to treatment with the one or more glycosaminoglycan (GAG)lyase.

In certain embodiments, methods and processes described herein areutilized to detect and/or measure an oligosaccharide (trisaccharide)having the formula:

[IdoA-GlcN(Ac)_(m)-IdoA](SO₃R)_(n)   Formula XXI

In certain embodiments, oligosaccharides described herein, e.g., thosedetected and/or measured according to the methods and/or processesdescribed herein, are trisaccharides of Formula XXI, wherein m is 0-1and n is 0-4. The trisaccharide is optionally sulfated with n SO₃Rgroups in any suitable location. In certain instances, compounds ofFormulas XXI are provided by treating the glycosaminoglycan heparansulfate with a suitable glycosaminoglycan lyase.

In certain embodiments, methods and processes described herein areutilized to detect and/or measure an oligosaccharide (trisaccharide)having the formula:

[IdoA-GlcN(Ac)_(m)-GlcA](SO₃R)_(n)   Formula XXII

In certain embodiments, oligosaccharides described herein, e.g., thosedetected and/or measured according to the methods and/or processesdescribed herein, are trisaccharides of Formula XXII, wherein m is 0-1and n is 0-3. The trisaccharide is optionally sulfated with n SO₃Rgroups in any suitable location. In certain instances, compounds ofFormulas XXII are provided by treating the glycosaminoglycan heparansulfate with a suitable glycosaminoglycan lyase.

In certain embodiments, methods and processes described herein areutilized to detect and/or measure an oligosaccharide (trisaccharide)having the formula:

[GlcA-GlcN(Ac)_(m)-GlcA](SO₃R)_(n)   Formula XXIII

In certain embodiments, oligosaccharides described herein, e.g., thosedetected and/or measured according to the methods and/or processesdescribed herein, are trisaccharides of Formula XXIII, wherein m is 0-1and n is 0-3. The trisaccharide is optionally sulfated with n SO₃Rgroups in any suitable location. In certain instances, compounds ofFormulas XXIII are provided by treating the glycosaminoglycan heparansulfate with a suitable glycosaminoglycan lyase.

In some embodiments, the detection and/or measurement of any one or moretrisaccharide of any of Formulas XXI to XXIII is utilized in a processfor diagnosing a disorder associated with the accumulation of heparansulfate (e.g., a heparan sulfate lysosomal storage disease such as, byway of non-limiting example, MPS I, MPS II, MPS IIIA, MPS IIIB, MPSIIIC, MPS IIID, MPS VII, or a combination thereof). In certainembodiments, an oligosaccharide of any of Formulas XXI to XXIII issaturated or unsaturated at the C4 and C5 positions of the saccharideresidue at the non-reducing end of the oligosaccharide. In someembodiments, a process for diagnosing a disorder associated with theaccumulation of heparan sulfate (e.g., MPS I, MPS II, MPS IIIA, MPSIIIB, MPS IIIC, MPS IIID, MPS VII, or a combination thereof) furthercomprises detecting one or more oligosaccharide, as set forth above andas appropriate (e.g., any of Formulas I-XX, as they pertain to theparticular disorder being diagnosed).

In certain embodiments, methods and processes described herein areutilized to detect and/or measure an oligosaccharide (trisaccharide)having the formula:

[IdoA-GalNAc-Ido](SO₃R)_(n)   Formula XXIV

In certain embodiments, oligosaccharides described herein, e.g., thosedetected and/or measured according to the methods and/or processesdescribed herein, are trisaccharides of Formula XXIV, wherein n is 0-4.The trisaccharide is optionally sulfated with n SO₃R groups in anysuitable location. In certain instances, compounds of Formulas XXIV areprovided by treating the glycosaminoglycan chondroitin and/or dermatansulfate with a suitable glycosaminoglycan lyase.

In certain embodiments, methods and processes described herein areutilized to detect and/or measure an oligosaccharide (trisaccharide)having the formula:

[IdoA-GalNAc-GlcA](SO₃R)_(n)   Formula XXV

In certain embodiments, oligosaccharides described herein, e.g., thosedetected and/or measured according to the methods and/or processesdescribed herein, are trisaccharides of Formula XXV, n is 0-3. Thetrisaccharide is optionally sulfated with n SO₃R groups in any suitablelocation. In certain instances, compounds of Formulas XXV are providedby treating the glycosaminoglycan chondroitin and/or dermatan sulfatewith a suitable glycosaminoglycan lyase.

In certain embodiments, methods and processes described herein areutilized to detect and/or measure an oligosaccharide (trisaccharide)having the formula:

[GlcA-GalNAc-Ido](SO₃R)_(n)   Formula XXVI

In certain embodiments, oligosaccharides described herein, e.g., thosedetected and/or measured according to the methods and/or processesdescribed herein, are trisaccharides of Formula XXVI, wherein n is 0-3.The trisaccharide is optionally sulfated with n SO₃R groups in anysuitable location. In certain instances, compounds of Formulas XXVI areprovided by treating the glycosaminoglycan chondroitin and/or dermatansulfate with a suitable glycosaminoglycan lyase.

In certain embodiments, methods and processes described herein areutilized to detect and/or measure an oligosaccharide (trisaccharide)having the formula:

[GlcA-GalNAc-GlcA](SO₃R)_(n)   Formula XXVII

In certain embodiments, oligosaccharides described herein, e.g., thosedetected and/or measured according to the methods and/or processesdescribed herein, are trisaccharides of Formula XXVII, wherein n is 0-2.The trisaccharide is optionally sulfated with n SO₃R groups in anysuitable location. In certain instances, compounds of Formulas XXVII areprovided by treating the glycosaminoglycan chondroitin and/or dermatansulfate with a suitable glycosaminoglycan lyase.

In some embodiments, the detection and/or measurement of any one or moretrisaccharide of any of Formulas XXIV to XXVII is utilized in a processfor diagnosing a disorder associated with the accumulation ofchondroitin and/or dermatan sulfate (e.g., a chondroitin and/or dermatansulfate lysosomal storage disease). In certain embodiments, anoligosaccharide of any of Formulas XXIV to XXVII is saturated orunsaturated at the C4 and C5 positions of the saccharide residue at thenon-reducing end of the oligosaccharide. In some embodiments, a processfor diagnosing a disorder associated with the accumulation ofchondroitin and/or dermatan sulfate further comprises detecting one ormore oligosaccharide, as set forth above and as appropriate (e.g., anyof Formulas I-XX, as they pertain to the particular disorder beingdiagnosed).

In certain embodiments, methods and processes described herein areutilized to detect and/or measure an oligosaccharide (trisaccharide)having the formula:

GlcA-GlcNAc-GlcA   Formula XXVIII

In certain embodiments, oligosaccharides described herein, e.g., thosedetected and/or measured according to the methods and/or processesdescribed herein, are trisaccharides of Formula XXVIII. In certaininstances, compounds of Formulas XXVIII are provided by treating theglycosaminoglycan hyaluronan with a suitable glycosaminoglycan lyase.

In some embodiments, the detection and/or measurement of a trisaccharideof Formula XXVIII is utilized in a process for diagnosing a disorderassociated with the accumulation of hyaluronan (e.g., a hyaluronanlysosomal storage disease). In certain embodiments, an oligosaccharideof Formula XXVIII is saturated or unsaturated at the C4 and C5 positionsof the saccharide residue at the non-reducing end of theoligosaccharide. In some embodiments, a process for diagnosing adisorder associated with the accumulation of hyaluronan furthercomprises detecting one or more oligosaccharide, as set forth above andas appropriate (e.g., any of Formulas I-XX, as they pertain to theparticular disorder being diagnosed).

In certain embodiments, methods and processes described herein areutilized to detect and/or measure an oligosaccharide (trisaccharide)having the formula:

[Gal-GlcNAc-Gal](SO₃R)_(n)   Formula XXIX

In certain embodiments, oligosaccharides described herein, e.g., thosedetected and/or measured according to the methods and/or processesdescribed herein, are trisaccharides of Formula XXIX, wherein n is 0-3.The trisaccharide is optionally sulfated with n SO₃R groups in anysuitable location. In certain instances, compounds of Formulas XXIX areprovided by treating the glycosaminoglycan keratan sulfate with asuitable glycosaminoglycan lyase.

In some embodiments, the detection and/or measurement of any one or moretrisaccharide of Formula XXIX is utilized in a process for diagnosing adisorder associated with the accumulation of keratan sulfate (e.g., akeratan sulfate lysosomal storage disease). In certain embodiments, anoligosaccharide of Formula XXIX is saturated or unsaturated at the C4and C5 positions of the saccharide residue at the non-reducing end ofthe oligosaccharide. In specific embodiments, the oligosaccharide issaturated at the C4 and C5 positions of the saccharide residue at thenon-reducing end of the oligosaccharide. In some embodiments, a processfor diagnosing a disorder associated with the accumulation of keratansulfate further comprises detecting one or more oligosaccharide, as setforth above and as appropriate (e.g., any of Formulas I-XX, as theypertain to the particular disorder being diagnosed).

In certain embodiments, methods and processes described herein areutilized to detect and/or measure an oligosaccharide (trisaccharide)having the formula:

[GalNAc6S-GlcA-(GalNAc)_(m)](SO₃R)_(n)   Formula XXX

In certain embodiments, oligosaccharides described herein, e.g., thosedetected and/or measured according to the methods and/or processesdescribed herein, are di- and tri-saccharides of Formula XXX, wherein nis 0-3. The di- or tri-saccharide is optionally sulfated with n SO₃Rgroups in any suitable location. In certain instances, compounds ofFormulas XXX are provided by treating the glycosaminoglycan chondroitinsulfate with a suitable glycosaminoglycan lyase. In some embodiments,the detection and/or measurement of any one or more disaccharide ortrisaccharide of Formula XXX is utilized in a process for diagnosing MPSIVA. In some embodiments, a di- or tri-saccharide of Formula XXX has astructure o as set forth in any of the disaccharides of FIG. 21.

In certain embodiments, methods and processes described herein areutilized to detect and/or measure an oligosaccharide (trisaccharide)having the formula:

[GalNAc6S-IdoA-(GalNAc)_(m)](SO₃R)_(n)   Formula XXXI

In certain embodiments, oligosaccharides described herein, e.g., thosedetected and/or measured according to the methods and/or processesdescribed herein, are di- or tri-saccharides of Formula XXXI, wherein nis 0-4 and m is 0-1. The di- or tri-saccharide is optionally sulfatedwith n SO₃R groups in any suitable location. In more specificembodiments, a disaccharide of Formula XVIII has a structure of any ofthe di- or tri-saccharides of FIG. 22. In certain instances, compoundsof Formulas XXXI-A to XXXI-T are provided by treating theglycosaminoglycan dermatan sulfate with a suitable glycosaminoglycanlyase.

In some embodiments, the detection and/or measurement of any one or moredi- or tri-saccharide of Formula XXXI is utilized in a process fordiagnosing MPS IVA.

In certain embodiments, methods and processes described herein areutilized to detect and/or measure an oligosaccharide (disaccharide)having the formula:

[GlcA-GalNAc](SO₃R)_(n)   Formula XXXII

In certain embodiments, oligosaccharides described herein, e.g., thosedetected and/or measured according to the methods and/or processesdescribed herein, are disaccharides of Formula XXXII, wherein n is 0-2.The disaccharide is optionally sulfated with n SO₃R groups in anysuitable location. In more specific embodiments, a disaccharide ofFormula XXXII has a structure as set forth in any of the disaccharidesof FIG. 23. In certain instances, compounds of Formulas XXXII-A toXXXII-C are provided by treating the glycosaminoglycan chondroitinsulfate with a suitable glycosaminoglycan lyase.

In some embodiments, the detection and/or measurement of any one or moredisaccharide of Formula XXXII is utilized in a process for diagnosingMPS VII.

In certain embodiments, an oligosaccharide of any of Formulas XXX toXXXII is saturated or unsaturated at the C4 and C5 positions of thesaccharide residue at the non-reducing end of the oligosaccharide.

In certain embodiments, any of the processes or methods provided hereincomprise detecting and/or measuring the amount of any oligosaccharideprovided in any of Formulas Ito XX, any of Formulas XXI to XXIX, or anyof Formulas Ito XXXII. In further embodiments, any process or methodprovided herein comprises detecting and/or measuring the amount of anyoligosaccharide provided in any of Formulas Ito XX, any of Formulas XXIto XXIX, or any of Formulas Ito XXXII, wherein the oligosaccharide isattached to a detectable label at the reducing end of theoligosaccharide.

In certain embodiments, provided herein is a compound of any of FormulasIto XX, any one of Formulas XXI to XXIX, or any of Formulas Ito XXXII.In some embodiments, provided herein is a compound of any of FIGS. 4-23.In certain embodiments, the compound of any of Formulas Ito XXXII orFIGS. 4-23 is an isolated and/or purified compound. In some embodiments,the isolated and/or purified compound of any of Formulas Ito XXXII orFIGS. 4-23 is substantially free of oligosaccharides comprising asaccharide residue at the non-reducing end of the oligosaccharide thatis unsaturated at the C4 and C5 positions. As an illustrative example ofnon-reducing end saccharide residues that are saturated and unsaturatedat the C4 and C5 positions, an L-iduronic acid (IdoA) residue that issaturated at the C4 and C5 positions has a structure as follows:

whereas an L-iduronic acid (IdoA) residue at the non-reducing end of theoligosaccharide that is unsaturated at the C4 and C5 positions may havea structure as follows:

or the like. Oligosaccharides having non-reducing end saccharideresidues that are saturated at the C4 and C5 position are referred toherein as “C4-C5 non-reducing end saturated oligosaccharides”.

In certain embodiments, unsaturated saccharide residues include theunsaturated residue of IdoA, which is denoted by

and the unsaturated residue of GlcA which is denoted by

In some embodiments, the isolated and/or purified compound describedherein comprises at least at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, at least 97%, at least 98%, or atleast 99% oligosaccharide (by weight). In further or alternativeembodiments, the isolated and/or purified compound comprises 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, at least 97%, at least 98%, or atleast 99% NRE-saturated oligosaccharide (by weight).

In some embodiments, provided herein is a composition comprising one ormore isolated oligosaccharide of any of FIGS. 4-23. In certainembodiments, the oligosaccharide present in the composition is less than90% by weight non-reducing end unsaturated oligosaccharide. In certainembodiments, the oligosaccharide present in the composition is less than80% by weight non-reducing end unsaturated oligosaccharide. In certainembodiments, the oligosaccharide present in the composition is less than70% by weight non-reducing end unsaturated oligosaccharide. In certainembodiments, the oligosaccharide present in the composition is less than60% by weight non-reducing end unsaturated oligosaccharide. In certainembodiments, the oligosaccharide present in the composition is less than50% by weight non-reducing end unsaturated oligosaccharide. In certainembodiments, the oligosaccharide present in the composition is less than40% by weight non-reducing end unsaturated oligosaccharide. In certainembodiments, the oligosaccharide present in the composition is less than30% by weight non-reducing end unsaturated oligosaccharide. In certainembodiments, the oligosaccharide present in the composition is less than25% by weight non-reducing end unsaturated oligosaccharide. In certainembodiments, the oligosaccharide present in the composition is less than20% by weight non-reducing end unsaturated oligosaccharide. In certainembodiments, the oligosaccharide present in the composition is less than15% by weight non-reducing end unsaturated oligosaccharide. In certainembodiments, the oligosaccharide present in the composition is less than10% by weight non-reducing end unsaturated oligosaccharide. In certainembodiments, the oligosaccharide present in the composition is less than5% by weight non-reducing end unsaturated oligosaccharide. In someembodiments, the composition further comprises chromatographic resin orelectrophoretic resin. In some embodiments, the resin is a highperformance liquid chromatographic resin, or the like.

The compounds of any of Formulas I-XXXII or FIGS. 4-23 are obtained inany suitable manner, e.g., by generation and purification from naturalsources, chemical synthesis, or any other suitable method. Thesestructures do not naturally exist in an isolated and/or purified form.

The described di- and trisaccharides are optionally generated andpurified from natural sources. In certain embodiments, starting withglycosaminoglycans from natural sources (for example, chondroitinsulfate, heparan sulfate, heparin, or synthetic heparin (or heparan)like carbohydrates), these di- and trisaccharides are generated bytreating the glycan with glycosaminoglycan lyases (for example heparinlyases or chondroitinases) and purifying the di- and trisaccharidesliberated from the pre-existing non-reducing ends. In specificinstances, the di- and trisaccharides liberated from the pre-existingnon-reducing end are unique because they do not contain a c4-5 doublebond due to the action of the lyase.

In certain embodiments, the yield of the desired di- and trisaccharidesare increased by using glycosaminoglycans that have been treated withglycan degradative enzymes (for example, heparanase or lysosomalexo-enzymes such as the 2-sulfatase, N-sulfatase, etc) to generatesmaller fragments or fragments with a greater number of desirablenon-reducing ends. In further or alternative embodiments, the yield isincreased by starting with glycosaminoglycans that have been isolatedfrom an organism or cell that has a defect in glycosaminoglycandegradation such that the desirable non-reducing ends are enriched (forexample, glycosaminoglycans from iduronidase deficient systems, would beenriched in glycosaminoglycans that terminal on the non-reducing endwith iduronate residues). As shown in FIGS. 24 and 25, these uniquenon-reducing end disaccharides can be isolated by HPLC. FIG. 24 showsdisaccharides isolated from heparan sulfate from a normal human sample,while FIG. 25 shows disaccharides isolated from an MPS I (iduronidasedeficient) sample. The saturated disaccharides (indicated with redarrows) can be isolated by collecting the appropriate fractions fromthis or a similar preparative chromatography.

Additional or alternative methods of obtaining such compounds include,e.g., chemical transformation of the unsaturated saccharides into thesaturated saccharides. The described di- and trisaccharides can besynthesized by chemical or chemoenzymatic methods. Chemical methods forthe synthesis of these saturated di- and trisaccharides are optionallyconverted from the methods described by Prabhu, Venot, and Boons(Organic Letters 2003 Vol. 5, No. 26 4975-4978), which is incorporatedherein for such synthesis.

In further embodiments, provided herein is a compound of any of FormulasI-XXXII or FIGS. 4-23, wherein the compound is tagged with any label asdescribed herein.

In some embodiments, an isolated or otherwise generated oligosaccharidedescribed herein has a molecular weight of less than 2000 g/mol, lessthan 1500 g/mol, less than 1000 g/mol, less than 500 g/mol, less than400 g/mol, less than 300 g/mol, less than 260 g/mol, less than 200g/mol, less than 100 g/mol, or the like (e.g., prior to tagging with anydetectable label that may be included in a process described herein).

Detection/Measurement Methods

Oligosaccharides (including e.g., oligosaccharides tagged withdetectable labels) described herein are detected and/or measured inprocesses described herein in any suitable manner. In certainembodiments, detectable labels are detected and/or quantified accordingto any process described herein using any technique, particularly anytechnique suitable for the detectable label utilized. In someembodiments, suitable detection techniques include, by way ofnon-limiting example, one or more of a mass spectrometer, a nuclearmagnetic resonance spectrometer, a UV-Vis spectrometer, an IRspectrometer, a fluorimeter, a phosphorimeter, a radiation spectrometer(e.g., a scintillation counter), a thin layer chromatographic technique,or the like. In certain embodiments, in any process described herein,oligosaccharides are optionally directly detected using a suitabletechnique, such as quantitative nuclear magnetic resonance. Quantitativenuclear magnetic resonance is also optionally utilized to quantifyand/or detect the presence of a detectable label. In certainembodiments, one or more oligosaccharides are optionally detected usinga suitable liquid chromatography mass spectrometer (LC-MS).

In some embodiments, oligosaccharides are tagged with an antibody orprobe, and are quantified using any suitable method (e.g., dot blottechniques, immune detection techniques (e.g., ELISA), or the like).

Various analytical methods useful for the processes described hereininclude, by way of non-limiting example, mass spectrometry,chromatography, HPLC, UPLC, TLC, GC, HPAEC-PAD,electrophoresis—capillary or gel, or the like. In certain embodiments,wherein a chromatographic technique is utilized, any suitable solventsystem is optionally employed. In certain embodiments, a column (e.g.,Cosmogel DEAE, Tsk Gel DEAE, Cosmogel QA, Cosmogel CM, Cosmogel SP, 130ABEH particle Phenyl (1.7, 2.5, 3.5, 5, or 10 uM particle size), 130A BEHparticle C18 (1.7, 2.5, 3.5, 5, or 10 uM particle size), HSS particleC18 (1.8, 3.5, or 5 uM particle size), 300A BEH particle C18 (1.7, 3.5,5, 10 uM particle size), or the like with suitable length and internaldiameter) is optionally loaded with an equilibrating solvent (e.g., abuffer or salt solution, such as a potassium acetate solution, sodiumchloride solution, sodium acetate solution, ammonium acetate solution,or the like), e.g., with a pH of about 6, 7, or 8. In some embodiments,the buffer or salt solution has a concentration of about 10 mM, 20 mM,30 mM, 50 mM, 100 mM, 500 mM, 1 M, 2 M, or the like. Any suitable flowrate is used, e.g., 0.5 mL/min, 1 mL, min, 1.5 mL/min, 2 mL/min, or thelike. Following equilibration, a linear gradient is optionally utilized.In some embodiments, the linear gradient is run over 1-20 min, 1-10 min,10-20 min, 1-5 min, 5-10 min, or the like. In certain embodiments, thegradient is a buffer or salt solution, e.g., as described above (e.g.,from 0 M to 0.5 M, from 0 M to 3 M, from 0.5 M to 2 M, from 0 M to 2 M,from 1 M to 2 M, from 0 M to 3 M, from 2 M to 0 M, from 3 M to 0 M, orthe like). Once the gradient has reached a final concentration, theeluent is optionally held at the final concentration for a suitableperiod of time (e.g., 1-20 min, 5-10 min, 10-15 min, 1-5 min, 1-10 min,15-20 min, or the like). After the optional holding of the finalconcentration, the eluent may be switched to a second solvent or solventsystem (e.g., an alcohol, such as methanol, ethanol, or isopropanol,acetonitrile, water, or the like). The switch to the second solventsystem may be over a period of time, e.g., 15 seconds, 30 seconds, 45seconds, 60 seconds, 2 min, 3 min, or the like. The second solventsystem is optionally held for a period of time, such as 1 min, 2 min, 3min, 4 min, 5 min, 6 min, or the like. Following the second solventsystem cycle, the column is optionally restored to initial solventconditions.

In certain embodiments, detection or measurement of an analytical deviceprovides for diagnosis of a disease, diagnosis of severity of a disease,of efficacy of a therapy, or analysis based on other processes describedherein. For example, in some embodiments, absence of a peak or signal(e.g., a peak or signal indicative of the presence of a particularoligosaccharide) indicates that an individual is in a non-diseasedstate, in remission for a disease state, or undergoing effective therapyfor a disease, depending on the circumstances of the diagnosis. Incertain embodiments, the presence and/or area of a peak or signal(including, e.g., the presence of a certain signal or peak pattern orfingerprint) is utilized to determine the severity of a disease. In someembodiments, the presence and/or area of a peak or signal is utilized todetermine disease, disease severity, disease carrier status or the like,based on a certain threshold value for the disease, disease severity,disease carrier status. Such thresholds are optionally determined in anysuitable manner, e.g., by analyzing control samples, such controlsamples coming from non-diseased individuals, diseased individuals, orthe like.

In certain embodiments, a control biological sample utilized in anyprocess described herein was provided from an individual that does notsuffer from a disorder being diagnosed. In other embodiments, a controlbiological sample is taken from an individual suffering from a disorderbeing diagnosed. In certain embodiments, the result obtained from thecontrol biological sample is stored in a database. In such cases a testsample is optionally compared to a plurality of control data in adatabase. Moreover in certain embodiments, any diagnostic processdescribed herein is optionally utilized alone or in combination withother diagnostic techniques. Other diagnostic techniques include, by wayof non-limiting example, symptom analysis, biopsies, detection ofaccumulation of other compounds in biological samples, or the like. Insome embodiments, control biological samples are optionally taken fromthe same individual at substantially the same time, simply from adifferent location (e.g., one inflamed/arthritic synovial joint fluid vsthe contralateral non-arthritic synovial joint). In other embodiments,control biological samples are optionally taken from the same individualat different points in time (e.g., before therapy and after therapy ifthe method being utilized is a method of monitoring a treatmenttherapy).

Detectable Labels

In the various embodiments of any process or method described herein,any suitable detectable label is optionally utilized. In someembodiments, detectable labels useful in the processes or methodsdescribed herein include, by way of non-limiting example, mass labels,antibodies, affinity labels, radiolabels, chromophores, fluorescentlabels, or the like.

Fluorescent labels suitable for use in various embodiments hereininclude, by way of non-limiting example, 2-aminopyridine (2-AP),2-aminobenzoic acid (2-AA), 2-aminobenzamide (2-AB), 2-aminoacridone(AMAC), p-aminobenzoic acid ethyl ester (ABEE), p-aminobenzonitrile(ABN), 2-amino-6-cyanoethylpyridine (ACP), 7-amino-4-methylcoumarine(AMC), 8-aminonaphthalene-1,3,6-trisulfate (ANTS),7-aminonaphthalene-1,3-disulfide (ANDS), and8-aminopyrene-1,3,6-trisulfate (APTS), or the like. The fluorescentlabels can be attached by reductive amination with the fluorescent labeland sodium cyanoborohydride or the like.

Mass labels suitable for use in various embodiments herein include, byway of non-limiting example, D-2-anthranilic acid, D-2-aminopyridine,D-methyl iodide, ¹³C methyl iodide, deuterated-pyridyl-amine, D-biotinor the like. The mass labels can be attached by permethylation orreductive amination by any method that is known to those of skill in theart.

Affinity labels suitable for use in various embodiments herein include,by way of non-limiting example, biotin and derivatives.

Radio labels suitable for use in various embodiments herein include, byway of non-limiting example, sodium borotritide (NaB³H₄), or the like.

Chromophores suitable for use in various embodiments herein include, byway of non-limiting example, 4-amino-1,1′-azobenzene,4′-N,N-dimethylamino-4-aminoazobenzene, aminoazobenzene,diaminoazobenzene, Direct Red 16, CI Acid Red 57, CI Acid Blue 45, CIAcid Blue 22, CL Mordant Brown 13, CI Direct Orange 75, or the like. Thechromophores may be labeled by any method that is known to those ofskill in the art, such as reductive amination with the chromophore andsodium cyanoborohydride.

In some embodiments, the detectable label is an antibody. In specificembodiments, the antibody is attached to a detectable compound, such asmass labels, radiolabels, chromophores, fluorescent labels, or the like.In some embodiments, antibodies are themselves detected and/or aredetectable in various manners, e.g., as a chromophore, a fluorophore, orthe like; or with a probe (e.g., using dot blot techniques,immune-detection techniques, or the like).

Purification Methods

In some embodiments, the processes described herein comprises furthertreatment steps of the test and/or control samples. For example, in someembodiments, the samples are homogenized and/or purified. In specificembodiments homogenization is achieved in any suitable manner including,by way of non-limiting example, with a basic solution (e.g., 0.1 NNaOH), sonication, tissue grinding or other chemical agents).

In certain embodiments, samples, including test samples and/or controlsamples, described herein are optionally purified prior toglycosaminoglycan processing (e.g., lyase treatment) and/orcharacterization. Test samples and/or control samples (i.e., one or moreor all of the glycans found therein) are optionally purified using anysuitable purification technique. Test samples and/or control samples areoptionally purified at any suitable point in a process described herein,including before or after tagging of the glycans founds within thesample. In certain embodiments, purification techniques includecentrifugation, electrophoresis, chromatography (e.g., silica gel oralumina column chromatography), gas chromatography, high performanceliquid chromatography (HPLC) (e.g., reverse phase HPLC on chiral orachiral columns), thin layer chromatography, ion exchangechromatography, gel chromatography (e.g., gel filtration or permeationor size exclusion chromatography, gel electrophoresis), molecular sievechromatography, affinity chromatography, size exclusion, filtration(e.g. through a florisil or activated charcoal plug), precipitation,osmosis, recrystallization, fluorous phase purification, distillation,extraction, chromatofocusing, supercritical fluid extraction,preparative flash chromatography (e.g., flash chromatography using aUV-Vis detector and/or a mass spectrometer (e.g., using the Biotage®suite of products) or the like.

In some embodiments, glycosaminoglycans, such as heparan sulfate, arenaturally found attached to a core protein (together forming aproteoglycan). In some embodiments, provided herein are purificationprocesses of separating glycosaminoglycan fragments (e.g., heparansulfate fragments) from proteoglycans prior to processing theglycosaminoglycans for detection.

Therapeutic Methods

Provided in certain embodiments are methods of treating disordersassociated with the abnormal degradation, biosynthesis and/oraccumulation of glycosaminoglycans (GAGs), the methods comprising:

-   -   a. administering an agent for treating MPS (e.g., an agent that        modulates (e.g., promotes and/or inhibits) glycosaminoglycan        biosynthesis and/or degradation) to an individual in need        thereof;    -   b. monitoring the accumulation of glycosaminoglycans in the        individual using any process described herein for detecting or        quantifying the amount of oligosaccharides (e.g., di- and/or        tri-saccharides, such as GAG oligosaccharides, including heparan        sulfate fragments) present in a lyase digested biological sample        (e.g., urine, serum, plasma, or CSF sample) according to any        process described herein.

Provided in further or alternative embodiments are methods of monitoringthe treatment of disorders associated with the abnormal degradation,biosynthesis and/or accumulation of glycosaminoglycans (GAGs), themethods comprising:

-   -   a. following administration of an agent for treating MPS (e.g.,        an agent that modulates (e.g., promotes and/or inhibits)        glycosaminoglycan biosynthesis and/or degradation) to an        individual in need thereof, using an analytical instrument to        detect the presence of and/or measure the amount of a population        of one or more C4-C5 non-reducing end saturated oligosaccharides        present in a transformed biological sample that has been        prepared by: treating a population of glycosaminoglycans, in or        isolated from a biological sample taken from the individual,        with at least one digesting glycosaminoglycan lyase to transform        the glycosaminoglycans into the population of the one or more        C4-C5 non-reducing end saturated oligosaccharide;    -   b. displaying or recording the presence of or a measure of a        population of one or more oligosaccharide.

In specific embodiments, the oligosaccharide(s) detected or measured isone or more C4-C5 non-reducing end saturated oligosaccharide(s). In someembodiments, the agent is administered one or more times. In certainembodiments, the agent is administered multiple times. In someembodiments, the agent is administered in a loading dose one or moretimes (e.g., in a loading dosing schedule) and subsequently administeredin a maintenance dose (e.g., in a maintenance dosing schedule, such asthree times a day, twice a day, once a day, once every two days, onceevery three days, once every four days, once a week, or the like). Insome embodiments, when GAG oligosaccharide accumulation begins toincrease or accelerate, the dose is optionally adjusted (e.g., themaintenance dose is increased, or an additional loading dose or dosingschedule is utilized).

In some embodiments, monitoring the accumulation of glycosaminoglycanscomprises repeating the step of: using an analytical instrument todetect the presence of and/or measure the amount of a population of oneor more oligosaccharides present in a transformed biological sample thathas been prepared by treating a population of glycosaminoglycans, in orisolated from a biological sample from the individual, with at least onedigesting glycosaminoglycan lyase to transform the glycosaminoglycansinto the population of the one or more oligosaccharide. In specificembodiments, the step is repeated at periodic intervals (e.g., everyday, every other day, every 2 days, every 3 days, every 4 days, everyweek, every month, every 3 months, quarterly, every 6 months, yearly, orthe like), at regular times following a dose (e.g., 4 hours after aadministration of the agent, 6 hours after administration of the agent,8 hours after administration of the agent, 12 hours after administrationof the agent, or the like), prior to administration of the dose (e.g.,immediately prior to administration of the agent, 2 hours prior toadministration of the agent, or the like), or any other monitoringschedule.

In some embodiments, the monitoring of the accumulation ofglycosaminoglycans is conducted over a period of time, e.g., over aweek, two weeks, a month, two months, three months, six months, a year,or the like. In some embodiments, the method for quantifying the amountof one or more oligosaccharides in a lyase digested biological sample(e.g., urine, serum, plasma, or CSF) comprises detecting and/ormeasuring (e.g., with an analytical device), one or moreoligosaccharides within the lyase digested biological sample from theindividual after the biological sample obtained from the individual hasbeen treated with one or more glycosaminoglycan lyases. In certainembodiments, such glycosaminoglycan lyases are suitable for preparingdi- and/or tri-saccharides from the glycosaminoglycans present in thebiological sample obtained from the individual. In certain instances,the reducing end of a representative portion of the one or moreoligosaccharides in the transformed biological sample is tagged with anysuitable detectable label (e.g., a mass label, a radio label, afluorescent label, a chromophore label, affinity label, an antibody). Insome embodiments, the process comprises displaying or recording such acharacterization of the population of oligosaccharides and/or taggedoligosaccharides.

In some embodiments, the agent that modulates glycosaminoglycanbiosynthesis includes glycosaminoglycan accumulation inhibitors, agentsthat promote glycosaminoglycan degradation, agents that activate enzymesthat degrade glycosaminoglycans, agents that inhibit biosynthesis ofglycosaminoglycans, or the like. In some embodiments, the agent thatmodulates glycosaminoglycan biosynthesis is an agent that selectivelymodulates heparan sulfate biosynthesis, an agent that selectivelymodulates chondroitin sulfate biosynthesis, an agent that selectivelymodulates dermatan sulfate biosynthesis, an agent that selectivelymodulates keratan sulfate biosynthesis, an agent that selectivelymodulates hyaluronan biosynthesis, or a combination thereof.

In some instances, the detection and/or the quantification of theidentity and/or amount of oligosaccharides present in a biologicalsample is used to identify and/or diagnose a disorder associated withabnormal degradation, biosynthesis and/or accumulation ofglycosaminoglycans in an individual suspected of having such a disorder.

In some instances, the detection and/or the quantification of theidentity and/or amount of oligosaccharides present in the biologicalsample is used to monitor severity and course of the disease in anindividual diagnosed with or suspected of having a disorder associatedwith the abnormal degradation, biosynthesis and/or accumulation ofglycosaminoglycans. In some instances, the detection and/or thequantification of the identity and/or amount of oligosaccharides presentin the biological sample is used to calculate the administered dose ofan agent that modulates (e.g., promotes and/or inhibits)glycosaminoglycan biosynthesis and/or degradation.

In certain instances, wherein following administration of a selecteddose of a an agent that modulates (e.g., promotes and/or inhibits)glycosaminoglycan biosynthesis and/or degradation, an individual'scondition does not improve, the detection and/or the quantification ofthe identity and/or amount of oligosaccharides present in a biologicalsample provides for a treatment regimen to be modified depending on theseverity and course of the disease, disorder or condition, previoustherapy, the individual's health status and response to the drugs, andthe judgment of the treating physician.

In certain embodiments, monitoring the accumulation ofglycosaminoglycans in the individual comprises detecting or quantifyingthe amount of an oligosaccharide (or one or more oligosaccharides) in asample obtained from the individual (e.g., according to any methoddescribed herein) to obtain a first accumulation result (e.g., aninitial reading before treatment has begun, or at any other time) and asecond accumulation result that is subsequent to obtaining the firstresult. In some embodiments, the second result is compared to the firstresult to determine if the treatment is effectively reducing,maintaining, or reducing the rate of increasing the oligosaccharidelevels in a substantially identically obtained sample from theindividual being treated. In certain embodiments, depending on thedifference between the first and second results, the treatment can bealtered, e.g., to increase or decrease the amount of agent administered;to substitute the therapeutic agent with an alternative therapeuticagent; or the like. In certain embodiments, the dose of the therapeuticagent is decreased to a maintenance level (e.g., if the oligosaccharidelevel has been reduced sufficiently); further monitoring ofoligosaccharide levels is optional in such situation, e.g., to ensurethat reduced or maintained levels of oligosaccharide (e.g., GAGoligosaccharide(s)) are achieved.

Alternatively, provided herein is a method of detecting response totherapy in an individual or a method of predicting response to therapyin an individual comprising:

-   -   a. administering an agent for treating MPS (e.g., an agent that        modulates (e.g., promotes and/or inhibits) glycosaminoglycan        biosynthesis and/or degradation) to a plurality of cells from an        individual in need thereof (e.g., a plurality of fibroblasts,        serum, plasma, or CSF cells from a human suffering from MPS);    -   b. monitoring the accumulation of glycosaminoglycans in the        plurality of cells using any process described herein for        detecting or quantifying the amount of oligosaccharides (e.g.,        di- and/or tri-saccharides, such as GAG oligosaccharides,        including heparan sulfate fragments) present in a lyase digested        biological sample from the plurality of cells according to any        process described herein.

In specific embodiments, the oligosaccharide(s) detected or measured isone or more C4-C5 non-reducing end saturated oligosaccharide(s). It isto be understood that a plurality of cells from an individual includescells that are directly taken from the individual, and/or cells that aretaken from an individual followed by culturing to expand the populationthereof.

As discussed above, FIGS. 24 and 25 illustrate that C4-C5 non-reducingend saturated oligosaccharides can be isolated by, e.g., HPLC.

In some embodiments, monitoring the accumulation of glycosaminoglycanscomprises repeating the step of: using an analytical instrument todetect the presence of and/or measure the amount of a population of oneor more oligosaccharides present in a transformed biological sample thathas been prepared by treating a population of glycosaminoglycans, in orisolated from a biological sample from the individual, with at least onedigesting glycosaminoglycan lyase to transform the glycosaminoglycansinto the population of the one or more oligosaccharide. In specificembodiments, the step is repeated at periodic intervals (e.g., everyday, every other day, every 2 days, every 3 days, every 4 days, everyweek, or the like), or any other monitoring schedule.

Disorders

Disorders associated with the abnormal degradation, biosynthesis and/oraccumulation of glycosaminoglycans useful in the treatment anddiagnostic methods and processes described herein include any disorderwherein accumulation of glycosaminoglycans and/or fragments thereof canbe detected in a biological sample taken from an individual sufferingfrom such a disorder. As discussed herein, disorders associated withabnormal glycosaminoglycan degradation, biosynthesis, and/oraccumulation include e.g., lysosomal storage diseases. In specificembodiments, a lysosomal storage disease is mucopolysaccharidosis (MPS).In some embodiments, a mucopolysaccharidosis (MPS) is MPS I, MPS II, MPSIIIA, MPS IIIB, MPS IIIC, MPS IIID, MPS IVA, MPS IVB, MPS VI, MPS VII,MPS IX, or a combination thereof.

In some instances, a lysosomal storage disease is caused by abnormalglycosaminoglycan degradation. In some instances, a lysosomal storagedisease causes an accumulation of heparan sulfate and/or dermatansulfate (e.g., MPS I) and is caused by an iduronidase deficiency. Insome instances, a lysosomal storage disease causes an accumulation ofheparan sulfate and/or dermatan sulfate (e.g., MPS II) and is caused bya 2-sulfatase deficiency. FIG. 26 illustrates accumulation ofoligosaccharides in MPS II human fibroblast versus the accumulation ofoligosaccharides in normal human fibroblasts. In some instances, alysosomal storage disease causes an accumulation of heparan sulfateand/or hyaluronan (e.g., MPS IIIB) and is caused by anN-acetylglulcosaminidase deficiency. In some instances, a lysosomalstorage disease causes an accumulation of heparan sulfate (e.g., MPSIIIC) and is caused by a glucosamine acetyltransferase deficiency. Insome instances, a lysosomal storage disease causes an accumulation ofheparan sulfate and/or keratan sulfate (e.g., MPS IIID) and is caused bya N-acetylglucosamine 6-sulfatase deficiency. In some instances, alysosomal storage disease causes an accumulation of keratan sulfate(e.g., MPS IVA) and is caused by a galactose 6-sulfatase deficiency. Insome instances, a lysosomal storage disease causes an accumulation ofkeratan sulfate (e.g., MPS IVB) and is caused by an N-acetylglactosamine4-sulfatase deficiency. In some instances, a lysosomal storage diseasecauses an accumulation of heparan sulfate and/or hyaluronan (e.g., MPSVII) and is caused by a glucuronidase deficiency. In some instances, alysosomal storage disease causes an accumulation of hyaluronan (e.g.,MPS IX) and is caused by a hyaluronidase deficiency.

In some embodiments, a MPS is Hunter's disease. In certain embodiments,Hunter's disease causes an accumulation of dermatan sulfate and heparansulfate glycosaminoglycans. In certain instances, the accumulation ofdermatan sulfate and heparan sulfate glycosaminoglycans in Hunter'sdisease is associated with a deficiency in a sulfatase. In someembodiments, the MPS is Hurler's disease. In certain instances, Hurler'sdisease causes an accumulation of dermatan sulfate and heparan sulfateglycosaminoglycans. In some instances, the accumulation of dermatansulfate and heparan sulfate glycosaminoglycans in Hurler's disease isassociated with a deficiency in an iduronidase.

In some embodiments, a disorder associated with abnormalglycosaminoglycan degradation, biosynthesis and/or accumulation isundesired angiogenesis (e.g., angiogenesis associated with cancer,diabetic blindness, age-related macular degeneration, rheumatoidarthritis, or psoriasis), insufficient angiogenesis (e.g., coronaryartery disease, stroke, or delayed wound healing), amyloidosis, a spinalcord injury, hypertriglyceridemia, inflammation, or a wound.

In some instances, amyloidosis is present in various diseases including,e.g., Alzheimer's disease, Parkinson's disease, type-2 diabetes,Huntington's disease, spongiform encephalopathies (Creutzfeld-Jakob,Kuru, Mad Cow), diabetic amyloidosis, rheumatoid arthritis, juvenilechronic arthritis, Ankylosing spondylitis, psoriasis, psoriaticarthritis, adult still disease, Becet syndrome, famalial Mediterraneanfever, Crohn's disease, leprosy, osteomyelitis, tuberculosis, chronicbronciectasis, Castleman disease, Hodgkin's disease, renal cellcarcinoma, carcinoma of the gut, lung or urogenital tract. In someinstances, the Alzheimer's disease is associated with changes in thecontent and structure of one or more GAG (e.g., heparan sulfate, keratansulfate, or the like).

In some embodiments, disorders associated with abnormalglycosaminoglycan accumulation include disorders associated withabnormal biosynthesis (e.g., polymerization and/or sulfation) ofglycosaminoglycans. In certain instances, the abnormal biosynthesis ofglycosaminoglycans results in glycosaminoglycans that are not readilydegraded by normal glycosaminoglycan degrading enzymes. In someinstances, disorders associated with abnormal GAG biosynthesis includeosteoarthritis. In certain instances, osteoarthritis is associated withchanges in sulfation of chondroitin sulfate, changes in length ofchondroitin sulfate, changes in expression levels of chondroitinsulfate, or any combination of thereof. In some instances,osteoarthritis is associated abnormal chondroitin sulfatesulfotransferase. In certain instances, the osteoarthritis is associatedwith changes in sulfation of dermatan sulfate, changes in length ofdermatan sulfate, changes in expression levels of dermatan sulfate, orany combination of thereof. In certain instances, the osteoarthritis isassociated with changes in sulfation of keratan sulfate, changes inlength of keratan sulfate, changes in expression levels of keratansulfate, or any combination of thereof.

In some embodiments, a disorder associated with abnormalglycosaminoglycan degradation, biosynthesis and/or accumulation ismacular corneal dystrophy. In some instances, macular corneal dystrophyis associated with low amounts of keratan sulfate. In more specificembodiments, the keratan sulfate levels are due to failure to initiatekeratan sulfate synthesis, polymerize the keratan sulfate chain length,or any combination thereof.

In some embodiments, a disorder associated with abnormalglycosaminoglycan degradation, biosynthesis and/or accumulation is aninfectious or viral disease. In some embodiments, the infectious orviral disease includes herpes, diphtheria, papilloma virus, hepatitis,HIV, coronavirus, or adenovirus.

In some embodiments, a disorder associated with abnormalglycosaminoglycan degradation, biosynthesis and/or accumulation is acancer. In certain embodiments, the cancer is breast cancer, ovariancancer, colorectal cancer, cervical cancer, pancreatic cancer, gastriccancer, esophageal cancer, head and neck cancer, hepatocellular cancer,prostate cancer, melanoma, osteosarcoma, endometrial cancer, multiplemyeloma, gastric cancer, lung cancer, glioma, kidney cancer, bladdercancer, thyroid cancer, neuroblastoma, or non-Hodgkin lymphoma.

In certain instances, cancer is associated with abnormal heparan sulfatedepolymerization and degradation that results in unbound, accumulatedheparan sulfate. In some instances, abnormal heparan sulfatedepolymerization and degradation is associated with melanomas, gliomas,multiple myelomas, ovarian, breast, colon, cervical, pancreatic,gastric, and esophageal cancers. In certain instances, abnormal heparansulfate depolymerization and degradation contributes to angiogenesis,metastasis and growth factor mobilization. In some instances, theabnormal heparan sulfate depolymerization and degradation is fromincreased activity of a heparanase.

In certain instances, cancer is associated with abnormal heparan sulfatesulfation that results in accumulated heparan sulfate. In someinstances, abnormal heparan sulfate sulfation is associated with coloncarcinoma, myeloma, ovarian cancer, hepatocellular carcinoma, head andneck squamous cell carcinoma and prostate cancer. In some instances,heparan sulfate sulfation is decreased in certain cancers, while inother embodiments; the heparan sulfate sulfation is increased. In someinstances, abnormal heparan sulfate sulfation is caused by abnormalheparan sulfate sulfotransferase function.

In certain instances, cancer is associated with abnormal chondroitinsulfate sulfation. In more specific embodiments, the abnormalchondroitin sulfate sulfation is associated with lung cancer. In someinstances, the chondroitin sulfate sulfation is increased in certaincancers. In certain instances, the abnormal chondroitin sulfatesulfation is caused by abnormal chondroitin sulfate sulfotransferasefunction. In some instances, increased production of chondroitin sulfateis associated in breast cancer, melanoma, and transformed fibroblasts.

In certain instances, cancer is associated with dermatan sulfateepimerase expression. In some instances, the dermatan sulfate epimeraseexpression is increased in squamous cell carcinoma, glioma,gynecological cancer, pancreatic cancer, colorectal carcinoma, andprostate cancer. In certain instances, the cancer is associated withaccumulation of dermatan sulfate levels. In some instances, the dermatansulfate levels are increased in pancreatic cancer.

In certain instances, cancer is associated with abnormal keratan sulfatesulfation. In some instances, the abnormal keratan sulfate sulfation isassociated with glioblastomas. In certain instances, abnormal keratansulfate sulfation is caused by abnormal keratan sulfate sulfotransferasefunction. In some instances, keratan sulfate expression is increased inglioblastomas.

In certain instances, cancer is associated with abnormal hyaluronanaccumulation. In some instances, abnormal hyaluronan accumulation isassociated with breast cancer, prostate cancer, aggressive subtypes ofnon-Hodgkin lymphoma, and colorectal cancer. In certain instances,hyaluronan accumulation contributes to metastasis of certain cancers. Insome instances, the hyaluronan accumulation results from the overexpression of a hyaluronan synthase.

Drug Screens

Provided in certain embodiments herein is a process for identifying anagent that inhibits the accumulation of glycosaminoglycans in a cell,the process comprising:

-   -   a. contacting a plurality of mammalian cells with a compound,        the plurality of mammalian cells being of a cell line that        accumulates an abnormal amount of glycosaminoglycans;    -   b. incubating the mammalian cells with the compound;    -   c. optionally transforming the mammalian cells e.g., by        isolating a population of glycosaminoglycans from the cells        (e.g., using any suitable method described herein);    -   d. contacting the mammalian cells and/or the isolated population        of glycosaminoglycans from step (c) with a GAG lyase (e.g.,        heparin lyase);    -   e. purifying a sub-population of oligosaccharides from step (d)        (e.g., using any suitable method described herein);    -   f. detecting the presence of and/or measuring the amount of one        or more oligosaccharides present in the sub-population (e.g.,        using LC-MS or GC-MS); and    -   g. displaying or recording a characterization of the        sub-population of one or more oligosaccharides.

In certain instances, the mammalian cells are optionally transformed bye.g., tagging a population of glycosaminoglycans on and/or in the cellswith a detectable label. In some instances, the mammalian cells areoptionally transformed by e.g., isolating a population ofglycosaminoglycans on and/or in the cells using any suitablepurification technique described herein.

In certain embodiments, the cell is present in an individual (e.g., ahuman or other mammal) and is incubated at body temperature. In someembodiments, the cell line that accumulates an abnormal amount ofglycosaminoglycans being a mucopolysaccharidosis (MPS) cell line (e.g.,a human MPS cell line). In more specific embodiments, the MPS cell lineis a cell line for MPS I, MPS II, MPS IIIA, MPS TIM, MPS IIIC, MPS IIID,MPS IVA, MPS IVB, MPS VI, MPS VII, MPS IX, or a combination thereof. Insome embodiments, the human MPS cell line is present in a human or othermammal. In some embodiments, inhibitors of the accumulation ofglycosaminoglycans are compounds that reduce the rate of accumulation ofglycosaminoglycans in the cell, and/or agents that reduce the totalamount of glycosaminoglycans accumulated in the cell (i.e., diminish theamount of glycosaminoglycan that has been accumulated in the cell).Agents that are optionally tested for the screening process describedherein include any compound such as, by way of non-limiting example, apolynucleotide (e.g., siRNA), a polypeptide, or a small moleculecompound (e.g., having a molecular weight of less than 2,000 g/mol).

EXAMPLES

Example 1—Purification: The biological sample (cells, tissue, blood,serum, or the like) is homogenized and solublized in 0.1-1.0 N NaOH(e.g., 0.1 N, 0.2 N, 0.3 N, 0.4 N, 0.5 N, 0.6 N, 0.7 N, 0.8 N, 0.9 N, or1.0 N) or acetic acid and then neutralized with acetic acid or NaOH.Next a small sample is taken to measure protein content of the sampleusing standard methods. 0.01-0.5 mg/mL (0.01 mg/mL, 0.07 mg/mL, 0.12mg/mL, 0.17 mg/mL, 0.22 mg/mL, 0.27 mg/mL, 0.32 mg/mL, 0.37 mg/mL, 0.42mg/mL, or 0.5 mg/mL) protease (trypsin, chymotrypsin, pepsin, pronase,papain, or elastase) is treated in 0.1-0.5 M (e.g., 0.1 M, 0.16 M, 0.23M, 0.32 M, 0.39 M, 0.44 M, or 0.5 M) NaCl, 0.01-0.1 M (e.g., 0.01 M,0.02 M, 0.04 M, 0.06 M, 0.08 M, 0.1 M) NaOAc, at pH 5.5-7.5 (e.g., 5.5,6.0, 6.5, 7.0, or 7.5) and 25-40 C (e.g., 25 C, 30 C, 35 C, or 40 C) for1-24 hours (e.g., 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 18 h, 24 h). The sampleis diluted to reduce the ionic strength and loaded onto an ion exchangecolumn in 5-100 mM (e.g., 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60mM, 70 mM, 75 mM, 80 mM, 90 mM, 95 mM, 100 mM) NaOAc pH 5-7 with 0-300mM NaCl. After washing, the bound glycosaminoglycans are eluted with5-100 mM NaOAc pH 5-7 (e.g., 5, 5.5, 6, 6.5, 7) with 0.8-3 M (e.g., 0.8M, 1 M, 1.2 M, 1.4 M, 1.6 M, 1.8 M, 2 M, 2.5 M, or 3 M) NaCl. The elutedglycans are then concentrated and desalted by ethanol precipitation,size exclusion, or other methods. The purified glycans are dried forfurther analysis.

Example 2-Digestion: Lyase digestion: The purified glycans areresuspended in 10-300 mM sodium acetate, tris, phosphate, or othersuitable buffer, 0.02-1 mM (e.g., 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) calcium acetate, pH 5-8 (e.g., 5,5.5, 6, 6.5, 7, 7.5, or 8), were digested with heparin lyases I, II,III, I and II, I and III, II and III, or I, II, and III (0.0.15-1.5milliunits of each in 100-ul reactions, IBEX, Montreal, Canada) at 25 to37° C. for 1 to 24 hours.

Example 3—Tagging: Dried glycan sample is re-suspended in 2-100 μL0.003-0.1 M (e.g., 0.003 M, 0.003 M, 0.03 M, 0.06 M, 0.1 M) AB, AA,AMAC, or Bodipy dye and incubated at room temperature for 1-120 minutes(e.g., 1-10 min, 10-15 min, 15-20 min, 20-25 min, 25-30 min, 30-40 min,40-50 min, 50-60 min, 60-90 min, 90-120 min). Next, the reaction isinitiated with 2-100 (2 μL, 5 μL, 10 μL, 15 μL, 20 μL, 25 μL, 30 μL, 40μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, or 100 μL) 1 M NaCNBH₄ and thereaction is allowed to proceed at 25-100 C. (e.g., 25 C, 30 C, 35 C, 40C, 50 C, 60 C, 70 C, 80 C, 90 C, 100 C).

Example 4—Detecting: HPLC separation of tagged saccharides was performedutilizing the following conditions:

-   -   Column types: 130A BEH particle Phenyl (1.7, 2.5, 3.5, 5, or 10        uM particle size), 130A BEH particle C18 (1.7, 2.5, 3.5, 5, or        10 uM particle size), HSS particle C18 (1.8, 3.5, or 5 uM        particle size), or 300A BEH particle C18 (1.7, 3.5, 5, 10 uM        particle size) with suitable length and internal diameter.    -   Buffer Conditions:        -   A=10 mM Ammonium Acetate with 0-20% methanol        -   B=100% Methanol        -   Initial Conditions: 70-95% A, 0-30% B    -   Flow Rate is constant at 0.05-1 ml/min    -   Runs a gradient down to 70-90% A, 10-30% B over 5-65 min.    -   At 8.1 min runs a gradient to 0-20% A, 80-100% B over 5-20 min.    -   5-65 min returns to initial conditions

Fluorescently tagged oligosaccharides (disaccharides) were detected atvarious elution times depending on the specific marker produced ofinterest. Elution order of unsaturated disaccharide standards was:

-   -   2SNS6S    -   NS6S    -   2SNS    -   NS    -   NA6S    -   NA

Example 5: Tested was the ability of a non-reducing end detection methodto identify and classify MPS samples by blindly analyzing normal and MPSGAGs from cultured fibroblasts. Human primary fibroblast cultures fromMPS patients with MPS I, II, IIA, and IIIB (GM01256, GM00298, GM00879,and GM01426, respectively) were obtained from the NIGMS Human GeneticCell Repository. Cells were seeded at high density and allowed toaccumulate GAGs for 7-14 days. At harvest, GAGs were isolated using theprocedure described in the specifications. Total GAGs were digested withheparin lyases to completion. A portion of the digested GAG fragmentswere analyzed by liquid chromatography tandem atmospheric pressureelectrospray mass spectrometry of aniline derivatized oligosaccharidesto determine if the diagnostic biomarkers were present. FIGS. 27, 28A,29A, 30A and 31A—Panel A shows representative structures of predictedoligosaccharides for each class of MPS. FIGS. 27, 28A, 29A, 30A and31A—Panels B, C, D, and E show the extracted ion currents forrepresentative biomarker(s) for MPS I (m/z=590.5−591.5+719.5−720.5), MPSII (m/z=719.5−720.5+799.5−800.5), MPS IIIA(m/z=831.5−832.5+960.5−961.5+1088.5−1089.5), and MPS IIIB(m/z=793.5−794.5+873.5−874.5+922.5−923.5+1002.5−1003.5+1130.5−1131.5),respectively. MS analysis was done according to the methods of Lawrenceet al. J Biol Chem. 2008 November 28;283(48):33674-84. Insets are thecorresponding MS spectra for the non-reducing end structures detected.By combining the known degradative defect with the mass spectrometrydata, the actual structures of the biomarkers can be deduced or reducedto a few likely structures. The sensitivity of the method to detect andcorrectly identify MPS disease from small cell culture samples indicatesthat this method has the sensitivity to work on clinically relevantsamples such as blood or urine.

Example 6: The non-reducing end biomarker method was used to measure theaccumulation of GAGs in the MPS IIIB mouse model. Previous reports havehad difficulty measuring GAG accumulation in the brain, despitemicroscopic validation of lysosomal inclusions. This has been especiallypuzzling because the primary phenotype in MPS IIIB patients areneurological symptoms. We used this challenging case to determine ifthis biomarker method can detect GAG accumulation in difficult tissues.20 mg (wet weight) tissue samples were homogenized and total cellularGAGs were purified using the method described in the specifications. Thepurified GAGs were them treated with heparin lyases to liberate theunique non-reducing ends from the heterogeneous collection of fragments.The presence of the MPS IIIB biomarkers {(dp3-1Ac, 2S), (dp3(1Ac, 3S),and (dp3(1Ac, 4S)} was analyzed by LC-MS. These studies verify that theGAG accumulation is occurring in the brain.

Example 7—Diagnosing Disease: The following data is generated bymeasuring the abundance of a saturated disaccharide liberated from GAGsby heparin lyases that accumulate in MPS I samples. Using the describedmethod, the analyzed saturated disaccharide elutes at 3.07 minutes.

Example 7A: In these studies, 9 μL frozen urine samples from MPS Iaffected dogs and carrier dogs (unaffected) were analyzed for thepresence of saturated disaccharide liberated from the non-reducing endof the disease causing glycans. Picomoles of saturated disaccharide permilliliter of serum are shown in FIG. 35. FIG. 35 illustrates that theassay is useful for detecting the presence of MPS disease in urinesamples.

Example 7B: In these studies 30 !IL frozen serum samples from MPS Iaffected dogs and carrier dogs (unaffected) were analyzed for thepresence of saturated disaccharide liberated from the non-reducing endof the disease causing glycans. Picomoles of saturated disaccharide permilliliter of serum are shown in FIG. 36. FIG. 36 illustrates that thisassay is useful for identifying the presence of MPS (e.g., MPS I)disease in serum. By using the unique markers appropriate for other MPSclasses, the same is accomplished for other MPS classes.

Example 7C: In these studies 30 μL frozen CSF samples from MPS Iaffected dogs and carrier dogs (unaffected) were analyzed for thepresence of saturated disaccharide liberated from the non-reducing endof the disease causing glycans. Picomoles of saturated disaccharide permilliliter of CSF are shown in FIG. 37. FIG. 37 illustrates that thismethod is useful for detecting disease in CSF from MPS patients.

Example 7D: To determine if the GAG accumulation assay described here isable to detect MPS I disease in tissue samples, we analyzed the GAGaccumulation in 2.5 mg samples of cortex taken from unaffected andaffected MPS I dogs. Samples were analyzed for the presence of saturateddisaccharide liberated from the non-reducing end of the disease causingglycans. Picomoles of saturated disaccharide per milligram of cortex areshown in FIG. 38. FIG. 38 illustrates this method is useful fordetecting MPS disease in tissue samples from patients.

Example 8—Diagnosing Severity of Disease: The following data isgenerated by measuring the abundance of a saturated disaccharideliberated from GAGs by heparin lyases that accumulate in MPS I samples.Using the described method, this analyzed saturated disaccharide elutesat 3.07 minutes.

In these studies 30 μL frozen serum samples from MPS I affected dogswere analyzed to measure the level of GAG accumulation in each dog.Because the MPS I dog model is a naturally occurring and geneticallyheterogeneous model, there is significant variation in disease severitybetween individual dogs. These differences in disease severity arerevealed by variations in the level of GAG accumulation between theindividual dogs using the methods described here. Samples were analyzedfor the presence of saturated disaccharide liberated from thenon-reducing end of the disease causing glycans. Picomoles of saturateddisaccharide per milliliter of serum are shown in FIG. 39. FIG. 39illustrates that this method can be used to detect the response totherapy in serum from MPS patients.

Example 9—Monitoring Response to Therapy: The following data isgenerated by measuring the abundance of a saturated disaccharideliberated from GAGs by heparin lyases that accumulate in MPS I samples.Using the described method, this analyzed saturated disaccharide elutesat 3.07 minutes.

Example 9A: In these studies 30 μL frozen CSF samples from MPS Iaffected dogs before treatment (untreated) and after monthly intrathecal(IT) treatment with laronidase (treated). Samples were analyzed for thepresence of saturated disaccharide liberated from the non-reducing endof the disease causing glycans. Picomoles of saturated disaccharide permilliliter of CSF are shown in FIG. 40. FIG. 40 illustrates that thismethod is useful for detecting the response to therapy in CSF from MPSpatients.

Example 9B: To determine if the GAG accumulation assay described here isable to measure different levels of response to treatments, a group ofMPS I dogs who were treated with IT laronidase were analyzed withrespect to their level of therapy directed antibodies. “Tolerant” dogsare dogs that do not mount a significant immune reaction to thelaronidase treatment while “not tolerant” dogs mount a significantantibody response. This antibody response is expected to reduce theefficacy of the enzyme replacement therapy, laronidase. To determine ifthe assay can detect this differential response, 30 μL frozen CSFsamples from MPS I affected dogs before treatment (untreated) and aftermonthly intrathecal (IT) treatment with 0.58 mg/kg laronidase (treated).Samples were analyzed for the presence of saturated disaccharideliberated from the non-reducing end of the disease causing glycans andsummarized with respect to their tolerance class. The response totherapy is shown for three different dogs affected with MPS I. Picomolesof saturated disaccharide per milliliter of CSF are shown in FIG. 41.FIG. 41 illustrates that this method is useful for detecting thedifferential response to therapy in CSF from MPS patients. In someembodiments, this process is used to guide the treatment for individualpatients.

Example 9C: To determine if the GAG accumulation assay described here isable to detect a response to therapy in MPS I disease in tissue samplestreated with IT laronidase, we analyzed the GAG accumulation in 2.5 mgsamples of cortex taken from unaffected and affected MPS I dogs.Differential response to therapy is seen based on the tolerance statusof the specific dogs. Samples were analyzed for the presence ofsaturated disaccharide liberated from the non-reducing end of thedisease causing glycans and summarized with respect to their toleranceclass. The response to therapy is shown for three different dogsaffected with MPS I. Picomoles of saturated disaccharide per milliliterof CSF are shown in FIG. 42. FIG. 42 illustrates that this method isuseful for detecting response to therapy in tissue samples frompatients.

Example 9D: In these studies 30 μL frozen serum samples from MPS Iaffected dogs before treatment (sample #1) and monthly samples throughweekly IV treatment with laronidase (samples #2, 3, 4 etc.). Serumsamples were taken immediately prior to the next IV infusion. Sampleswere analyzed for the presence of unsaturated disaccharides liberatedfrom the non-reducing end of the disease causing glycans. The responseto therapy is shown for three different dogs affected with MPS I.Picomoles of these structures per milliliter of serum are shown in FIG.43A, 43B, and 43C. FIG. 43A, 43B, and 43C illustrate that this method isuseful for detecting the response to therapy in serum from MPS patients.

Example 10—Diagnosing Carriers: To determine if the GAG accumulationassay described here is able to detect carriers of the genetic cause ofMPS, we analyzed wild-type dogs and dogs that are heterozygous for theMPS I causing mutation. Carriers are presumed to lack any clinicalmanifestation of MPS I; however, it is possible that the partialdeficiency in a lysosomal enzyme will lead to low levels of GAGaccumulation that can be detected with this assay. Samples were analyzedfor the presence of unsaturated saturated disaccharide liberated fromthe non-reducing end of the disease causing glycans and summarized withrespect to their tolerance class. Picomoles of saturated disaccharideper milligram of tissue are shown in FIG. 44. FIG. 44 illustrates thatthe method described herein is useful for detecting carriers of thegenetic cause of MPS.

1.-30. (canceled)
 31. A method for determining in an individual, thepresence, identity, and/or severity of MPS I disorder, the methodcomprising: a) generating a biomarker comprising of one or morenon-reducing end oligosaccharides, wherein the biomarker is generated bytreating a population of glycosaminoglycans, in or isolated from abiological sample from the individual, with at least one digestingglycosaminoglycan lyase, wherein prior to lyase treatment, the biomarkeris not present in abundance in samples from individuals with the MPS Idisorder relative to individuals without the MPS I disorder; and b)using an analytical instrument to detect the presence of and/or measurethe amount of the biomarker produced and displaying or recording thepresence of or the measure of the biomarker produced; wherein thepresence and/or the measure of the amount of the biomarker are utilizedto determine the presence, identity, and/or severity of MPS I disorder;and wherein the biomarker is selected from a group consisting of FormulaI-A: IdoA-GlcNAc; Formula I-B: IdoA-GlcNS; Formula I-C: IdoA-GlcNS6S;Formula XII: [IdoA-GalNAc](SO₃R)_(n), wherein n is 0-2; Formula XXI:[IdoA-GlcN(Ac)m-IdoA](SO₃R)_(n), where m is 0-1 and n is 0-4; FormulaXXII: [IdoA-GlcN(Ac)m-GlcA](SO₃R)_(n), where m is 0-1 and n is 0-3; andFormula XXIII: [GlcA-GlcN(Ac)m-GlcA](SO₃R)_(n), where m is 0-1 and n is0-3.
 32. The method of claim 31, wherein the biomarker is selected fromthe group consisting of: Formula I-A: IdoA-GlcNAc; Formula I-B:IdoA-GlcNS; Formula I-C: IdoA-GlcNS6S; and Formula XII:[IdoA-GalNAc](SO3R)n, wherein n is 0-2.
 33. The method of claim 31,wherein the biomarker is selected from the group of consisting of:Formula I-A: IdoA-GlcNAc, Formula I-B: IdoA-G1cNS, and Formula I-C:IdoA-GlcNS6S.
 34. The method of claim 31, wherein the biomarker is ofFormula I-A: IdoA-GlcNAc.
 35. The method of claim 31, wherein thebiomarker is of Formula I-B: IdoA-GlcNS.
 36. The method of claim 31,wherein the biomarker is of Formula I-C: IdoA-GlcNS6S.
 37. The method ofclaim 31, wherein the biomarker is of formula XII selected from thegroup consisting of IdoA-GalNAc, IdoA-GalNAc6S, IdoA-GalNAc4S, andIdoA-GalNAc4 S6 S.
 38. The method of claim 31, wherein the methodfurther comprises purifying the biomarker generated in step (a) to yieldan isolated population of biomarkers.
 39. The method of claim 38,wherein the biomarker is purified using chromatography orelectrophoresis.
 40. The method of claim 31, wherein the method furthercomprises tagging the biomarkers with a detectable label.
 41. The methodof claim 40, wherein the detectable label is a mass label, a radiolabel, a fluorescent label, a chromophore label, or affinity label. 42.A method for determining response to a therapy in an individual havingan MPS I disorder, the method comprising: (a) generating a biomarkercomprising one or more saturated non-reducing end oligosaccharides,wherein the biomarker is generated by treating a population of apopulation of glycosaminoglycans, in or isolated from a biologicalsample from the individual, with at least one digestingglycosaminoglycan lyase, wherein prior to lyase treatment, the biomarkeris not present in abundance in samples from individuals with the MPS Idisorder relative to individuals without the MPS I disorder; and (b)using an analytical instrument to detect the presence of and/or measurethe amount of the biomarker produced and displaying or recording thepresence of or the measure of the biomarker produced; wherein thepresence of and/or measure of the amounts of the biomarker are utilizedto monitor the treatment of the MPS I disorder; and wherein thebiomarker is selected from a group consisting of: Formula I-A:IdoA-GlcNAc; Formula I-B: IdoA-GlcNS; Formula I-C: IdoA-GlcNS6S; FormulaXII: [IdoA-GalNAc](SO₃R)_(n), wherein n is 0-2; Formula XXI:[IdoA-GlcN(Ac)m-IdoA](SO₃R)_(n), where m is 0-1 and n is 0-4; FormulaXXII: [IdoA-GlcN(Ac)m-GlcA](SO₃R)_(n), where m is 0-1 and n is 0-3; andFormula XXIII: [GlcA-GlcN(Ac)m-GlcA](SO₃R)_(n), where m is 0-1 and n is0-3.
 43. The method of claim 42, wherein the biomarker is of FormulaI-A: IdoA-GlcNAc.
 44. The method of claim 42, wherein the biomarker isof Formula I-B: IdoA-GlcNS.
 45. The method of claim 42, wherein thebiomarker is of Formula I-C: IdoA-GlcNS6S.
 46. The method of claim 42,wherein the biomarker is of formula XII selected from the groupconsisting of IdoA-GalNAc, IdoA-GalNAc6S, IdoA-GalNAc4S, andIdoA-GalNAc4 S6 S.
 47. The method of claim 42, wherein the methodfurther comprises purifying the biomarker generated in step (a) to yieldan isolated population of biomarkers.
 48. The method of claim 47,wherein the biomarker is purified using chromatography orelectrophoresis.
 49. The method of claim 42, wherein the method furthercomprises tagging the biomarkers with a detectable label.
 50. The methodof claim 49, wherein the detectable label is a mass label, a radiolabel, a fluorescent label, a chromophore label, or affinity label.